UNIVERSITY  FARM 


MEDICAL  BACTERIOLOGY 


RODDY 


MEDICAL 
BACTERIOLOGY 


BY 

JOHN  A.   RODDY,  M.  D. 

ASSOCIATE   IN   HYGIENE    AND  BACTERIOLOGY,  JEFFERSON   MEDICAL   COLLEGE;  CHIEF 

ASSISTANT,   DEPARTMENT    OF    CLINICAL    MEDICINE,   JEFFERSON   HOSPITAL; 

PROFESSOR     OF     HYGIENE     AND     BACTERIOLOGY,     PHILADELPHIA 

COLLEGE   OF  PHARMACY;  SOMETIME   SEROLOGIST  TO  THE 

PHILADELPHIA   GENERAL   HOSPITAL',    MAJOR, 

MEDICAL   SECTION   O.  R.  C.,  U.  S.  A. 


WITH  46  ILLUSTRATIONS  OF 
WHICH  8  ARE  PRINTED  IN  COLORS 


PHILADELPHIA 

P.   BLAKISTON'S  SON  &  CO, 

1012  WALNUT  STREET 


COPYRIGHT,  1917,  BY  P.  BLAKTSTON'S  SON  &  Co. 


MAPLE    PRESS    YORK    PA 


PREFACE 


The  more  intimate  one's  knowledge  of  general  bacteriology  and  its  special 
subdivisions,  the  better  is  he  equipped  to  engage  in  any  particular  branch  of 
the  work.  Some  familiarity  with  physics  and  chemistry  is  essential  and  the 
extent  of  one's  knowledge  in  these  allied  sciences  largely  determines  the  extent 
to  which  one  can  cope  with  bacteriological  problems. 

This  subject  has  so  expanded  that  a  complete  systematic  exposition  of  it  is 
no  longer  possible  in  a  single  volume,  and  the  frequency  with  which  valuable 
new  facts  are  disclosed,  and  new  tests  and  technique  devised  makes  it  impera- 
tive for  one  engaged  exclusively  in  bacteriological  work  to  read  the  current 
journals  devoted  to  it. 

The  application  of  bacteriology  to  the  solution  of  many  important  problems 
arising  in  medicine,  veterinary  surgery,  agriculture  and  industry  has  created  a 
demand  that  some  knowledge  of  this  science  be  possessed  by  those  engaged  in 
diverse  occupations. 

There  are  basic  facts  and  technique  common  to  all  branches  of  bacteriology 
and  in  each  specialty  a  few  procedures  of  prime  importance.  A  knowledge  of 
these  is  the  first  requirement  of  all  beginners;  this  alone  constitutes  the  most 
valuable  information  on  the  subject  that  can  be  imparted  in  medical  and  other 
general  courses  of  instruction.  It  is  also  the  foundation  necessary  for  those  who 
intend  to  devote  themselves  exclusively  to  bacteriology,  and  for  further  inde- 
pendent work  in  this  field. 

The  author,  being  cognizant  of  the  needs  of  students,  practitioners  of  medi- 
cine, pharmacists  and  those  engaged  in  the  foodstuff  industries,  and  having  the 
invaluable  advice  and  guidance  of  Prof.  R.  C.  Rosenberger  and  the  able  assist- 
ance of  Dr.  Louis  Gershenfeld,  has  endeavored  to  present  in  the  clearest  form 
a  text  book  for  beginners  and  laboratory  guide  for  medical  practitioners  and 
pharmacists. 

It  is  a  pleasure  to  acknowledge  the  invaluable  assistance  rendered  by  Dr. 
Robert  M.  Lukens,  who  made  the  illustrations,  Mr.  David  R.  Brewer,  Dr. 
M.  E.  Smoczynski  and  Mrs.  Mary  L.  Vogel. 


CONTENTS 


PART  I 

CHAPTER  I 

PACK 

Introduction • I 

CHAPTER  II 
Classification  of  Bacteria 4 

CHAPTER  III 
Microscopy 9 

CHAPTER  IV 
Staining 15 

CHAPTER  V 
Sterilization 23 

CHAPTER  VI 
Culture  Media 40 

CHAPTER  VII 

Staphylococci 55 

CHAPTER  VIII 
Streptococci 59 

CHAPTER  IX 

Pneumococcus  (Diplococcus  Lanceolatus) 63 

CHAPTER  X 
Meningococcus 68 

CHAPTER  XI 

Gonococcus 71 

vii 


Vlll  CONTENTS 

CHAPTER  XII 

PAOB 
Micrococcus  Catarrhalis 74 

CHAPTER  XIII 
Micrococcus  Tetragenus  and  Sarcina 75 

CHAPTER  XIV 

Bacillus  of  Influenza,  Koch  Week's  Bacillus,  and  Bordet-Gengou  Bacillus 77 

CHAPTER  XV 
Bacillus  of  Morax  and  Axenfeld 79 

CHAPTER  XVI 
Pneumobacillus  or  Bacillus  of  Friedlander  (Bacillus  Mucosus  Capsulatus)     ....      80 

CHAPTER  XVII 
Diphtheria  Bacillus  (Klebs-Loeffler  Bacillus) 82 

CHAPTER  XVIII 
Pseudo-Diphtheria  Bacillus;  Hoffman's  Bacillus;  Xerosis  Bacillus 89 

CHAPTER  XIX 
Bacillus  and  Spirillum  of  Vincent 91 

CHAPTER  XX 
Tubercle  Bacillus 94 

CHAPTER  XXI 
Bacillus  of  Leprosy,  The  Smegma  and  Other  Acid- Fast  Bacteria 101 

CHAPTER  XXII 

The  Colon  Bacillus 103 

CHAPTER  XXIII 
The  Typhoid  Bacillus 106 

CHAPTER  XXIV 
The  Paratyphoid  and  Similar  Bacilli 109 

CHAPTER  XXV 
Diagnosis  of  Typhoid  Fever  and  Food  Poisoning  Infections Ill 


CONTENTS  IX 

CHAPTER  XXVI 

PAGE 

Dysentery  Bacilli 119 

CHAPTER  XXVII 
Spirillum  Cholerae  Asiaticae,  Vibrio  Cholerae  Asiaticae  (Comma  Bacillus) 122 

CHAPTER  XXVIII 
Micrococcus  Melitensis 125 

CHAPTER  XXIX 

Bacillus  Pyocyaneus 126 

CHAPTER  XXX 
Bacillus  Proteus  Vulgaris 128 

CHAPTER  XXXI 
Bacillus  Lactis  Aerogenes,  Bacillus  Bulgaricus 129 

CHAPTER  XXXII 
Bacillus  Botulinus 13° 

CHAPTER  XXXIII 
Bacillus  Aerogenes  Capsulatus 132 

CHAPTER  XXXIV 
Bacillus  Maligni  (Edematis 134 

CHAPTER  XXXV 
Bacillus  Tetani 136 

CHAPTER  XXXVI 
Bacillus  Anthracis 139 

CHAPTER  XXXVII 

Bacillus  Subtilis 141 

CHAPTER  XXXVIII 
Bacillus  Prodigiosus      143 

CHAPTER  XXXIX 
Bacillus  Pestis 144 


X  CONTENTS 

CHAPTER  XL 

PAGE 
Bacillus  Mallei I47 

CHAPTER  XLI 

Spirochaeta,  Obermeyeri I.g 

CHAPTER  XLII 

Treponema  Pallidum .150 

CHAPTER  XLIII 

Spirochaeta  Pertenuis j,.. 

CHAPTER  XLIV 

The  Higher  Bacteria I5,- 

CHAPTER  XLV 

Hyphomycetes  (Molds) l6l 

CHAPTER  XLVI 

Saccharomycetes I^. 

CHAPTER  XLVII 

Monila l6; 

CHAPTER  XLVIII 

Sporotrichum  Schenkii .168 

CHAPTER  XLIX 
Infectious  Diseases  of  Unknown  Causation 


PART   11 

CHAPTER  I 
Examination  of  Water 177 

CHAPTER  II 
Examination  of  Milk £76 


CONTENTS  XI 

CHAPTER  III 

PAGE 

Examination  of  Fluids  and  Solids 178 

CHAPTER  IV 
Determination  of  the  Germicidal  Power  of  Chemical  Disinfectants.    .......    185 

CHAPTER  V 

Diagnosis 2O1 

CHAPTER  VI 

Bacterial  Vaccines 2I1 

CHAPTER  VII 
Therapeutic  Sera 223 

CHAPTER  VIII 

Wassermann  and  Other  Complement  Fixation  Tests 226 

CHAPTER  IX 

Immunity 257 

Index  .  279 


MEDICAL    BACTERIOLOGY 


PART  I 

CHAPTER  I 

The  first  intimation  that  disease  might  be  due  to  minute  organisms  was  given 
by  Kircher,  a  member  of  the  Society  of  Jesus,  in  1646.  He  reported  the  pres- 
ence of  "minute  living  worms"  in  putrid  meat,  attributed  the  putrefaction  to 
their  activity  and  suggested  that  disease  might  be  due  to  similar  organisms. 

Twenty-five  years  later  he  reached  the  conclusion  that  puerperal  fever  was 
caused  by  animalculae  (bacteria).  In  1658  Hook  made  similar  observations 
and  confirmed  Kircher's  work. 

Leeuwenhoek,  a  linen  draper,  who  entered  his  trade  in  childhood  and  lacked 
schooling,  studied  lens  making,  devised  lenses  much  superior  to  any  previously 
made,  and  so  opened  up  vast  possibilities.  With  his  improved  instruments,  he 
observed  animalculae  in  saliva,  feces  and  vegetable  infusions.  His  observations 
were  reported  in  1660. 

At  that  time  the  discoveries  of  these  men  excited  some  interest,  but  it  was 
not  general  and  advancement  lagged  for  more  than  a  half  a  century.  Then  in 
1762  Plenciz  confirmed  the  observations  of  Kircher  and  Leeuwenhoek  and 
assumed  a  relationship  between  animalcules  and  disease  in  general. 

Spallanzani,  in  1777,  demonstrated  that  boiling  and  hermetically  sealing 
infusions  prevented  fermentation. 

Microscopic  observations  were  becoming  more  scientific  and  in  1786  Muller 
divided  animalculae  into  two  classes — monas  and  vibrio. 

At  the  close  of  the  eighteenth  century  (1798)  another  great  advance  was 
made  when  Jenner  discovered  the  method  of  immunizing  against  small  pox, 
a  method  that  is  still  in  vogue. 

In  1835  Bassi  reported  certain  diseases  of  silkworms  due  to  the  activity  of 
fungi.  In  1840  Henle  postulated  that  which  is  commonly  referred  to  as  "  Koch's 
Law:" 

I.  A  specific  microorganism  must  be  constantly  associated  with  the  disease  * 

II.  It  must  be  isolated  and  studied  apart  from  the  disease. 

III.  When  introduced  into  healthy  animals  it  must  produce  the  disease, 
and  in  the  animal  in  which  the  disease  has  been  produced  experimentally,  the 
organisms  must  be  found  under  the  original  conditions. 

Semmelweiss,  of  Vienna,  a  brilliant  martyr,  sacrificed  by  the  bigotry  of  his 
profession,  in  1847  reached  the  conclusion  that  the  cause  of  puerperal  fever  was 


2  MEDICAL  BACTERIOLOGY 

transmitted  from  the  dissecting  room  to  patients  by  students'  fingers,  and  to 
curtail  its  occurrence  advised  students  to  wash  their  hands  prior  to  making 
vaginal  examinations.  For  this  innovation  the  elect  ostracized  him. 

In  1850  Rayer  and  Davine  discovered  a  bacterium  in  the  blood  of  animals 
afflicted  with  splenic  fever.  Thirteen  years  later  Davine  disclosed  the  relation 
of  bacillus  anthrax  to  splenic  fever. 

Until  this  time  microorganisms  associated  with  putrefaction  and  disease  had 
been  classified  as  worms  or  animal  organisms;  Robin,  in  1853,  classified  them 
with  the  genus  leptothrix,  as  belonging  to  the  algae.  In  1859  Davine  studied 
the  subject  in  a  broader  way  than  had  been  done  before<  and  insisted  upon  their 
vegetable  nature.  Later  Colm  confirmed  these  contentions  and  bacteria  have 
since  been  generally  considered  as  vegetable  organisms. 

By  the  middle  of  the  nineteenth  century  the  belief  in  a  relationship  of  bac- 
teria to  disease  was  beginning  to  affect  medical  thought,  as  evidenced  in  Henle's 
"Text  Book  of  Rational  Pathology,"  published  in  1853.  Reinfleisch  declared 
wound  infection  was  due  to  microbic  invasion.  In  1866,  two  years  later,  Walder 
expressed  the  same  belief. 

Regardless  of  the  many  observations,  the  collection  of  considerable  evidence 
and  the  presentation  of  many  hypotheses,  some  of  which  were  surprisingly 
accurate,  the  true  relationship  of  bacteria  to  life  and  disease  was  not  appreciated 
until  after  the  publication,  by  Pasteur,  in  1869,  of  the  first  complete  study  of  a 
contagious  disease  caused  by  microorganisms.  To  this  man  and  the  country 
which  produced  him  and  facilitated  his  investigations,  the  world  is  indebted  for 
the  study  and  presentation  of  the  subject  in  such  a  way  as  to  awaken  general 
interest,  and  bring  forth  untold  blessings. 

About  this  same  time  Koch  introduced  the  cultivation  of  bacteria  on  solid 
culture  media  and  in  many  ways  greatly  expanded  the  possibilities  of  bacterio- 
logical studies. 

From  that  time  on  great  discoveries  followed  rapidly,  one  after  the  other, 
most  of  them  arrived  at  after  years  of  study  and  research,  few  by  accident. 

In  1875,  Lister  inaugurated  antiseptic  surgery,  an  epoch  in  that  art. 

Ehrlich  and  Weigert  introduced  staining  methods  for  the  study  of  bacteria 
in  1877.  In  1879  Frank  began  the  study  of  the  relationship  of  bacteria  to  the 
growth  of  leguminous  roots. 

The  year  1884  may  be  considered  the  golden  year  of  bacteriology;  among 
other  advances  made  may  be  noted  the  discovery  of  the  diphtheria  bacillus  by 
Klebs  and  LoerHer,  the  pneumococcus  by  Fraenkel,  the  tetanus  bacillus  by 
Nicolaier,  Gaffky's  extended  studies  of  the  typhoid  bacillus  (discovered  four 
years  earlier  by  Eberth),  the  discovery  by  Pasteur  of  his  method  of  immunizing 
against  rabies,  and  the  discovery  by  Koch  of  the  spirillum  of  cholera. 

A  great  stride  forward  was  taken  in  1890  when  Behring  introduced  diphtheria 
antitoxin. 

The  discovery  of  the  specific  cause  of  syphilis  by  Schaudin  in  1905,  the  intro-' 
duction  of  opsonic  therapy  by  Wright  in  1907,  and  the  antimeningococcus  serum 
of  Flexner,are  but  a  few  of  the  recent  advances  in  this  rapidly  developing  science. 


MEDICAL  BACTERIOLOGY  3 

The  most  notable  recent  event  in  the  history  of  American  bacteriology  was 
the  publication  of  the  first  number  of  the  Journal  of  Bacteriology,  January, 
1916,  the  first  American  journal  devoted  exclusively  to  this  science.' 

A  more  complete  and  interesting  presentation  of  the  history  of  bacteriology 
may  be  found  in. the  following  books: 

"  Vaccination,  Its  Natural  History  and  Pathology,"  S.  M.  Copeman. 

"  History  of  The  Plague  in  London,"  Daniel  DeFoe. 

"  Infection  and  Parasitic  Diseases  Including  Their  Causes  and  Manner  of 
Transmission,"  Millard  Langfeld. 

"Bacteria,"  George  Newman. 

"Scientific  Features  of  Modern  Medicine,"  Frederic  S.  Lee. 

"History  of  Medicine,"  Fielding  H.  Garrison. 

"History  of  Medicine,"  J.  H.  Baas,  translated  by  Handerson. 


CHAPTER  II 
CLASSIFICATION 

Bacteria  have  some  properties  allied  to  the  lower  forms  of  animal  life — 
flagellata — but  their  closest  relationship  is  to  the  lower  algae.  They  are  gen- 
erally considered  to  be  vegetable  organisms  and  are  classified  as  fission  fungi  or 
schizomycetes.  They  are  microscopic,  unicellular  organisms  which  propagate 
by  cleavage  or  fission.  Of  the  various  systems  of  classification  of  bacteria,  none 
of  which  is  complete  or  satisfactory,  the  one  most  commonly  adopted  by  writers 
is  the  following  by  Migula. 

CLASSIFICATION  OF  BACTERIA 

Coccaceae. — Spherical  forms,  with  divisions  in  one  to  three  planes,  forming 
two  to  eight  daughter  cells. 

Streptococcus. — Cocci  without  flagella,  dividing  in  one  plane,  often  remain- 
ing connected  so  as  to  form  beaded  chains. 
Micrococcus. — Without  flagella  dividing  in  two  planes.     When  remaining 

connected,  after  fission,  they  form  grape-like  clusters  (staphylococci). 
Sarcina. — Without  flagella,  dividing  in  three  planes.     Usually  remaining 

connected  to  form  cubical  packets. 

Planococcus. — Possessing  flagella,  dividing  in  two  planes,  sometimes  re- 
maining connected  to  form  sheets. 

Piano sarcina. — Possessing  flagella,  dividing  in  three  planes, 
Bacteriaceae. — Elongated  straight  cells  dividing  in  the  transverse  plane  only. 
Bacterium. — Without  flagella,  sometimes  with  endospores. 
Bacillus. — With  flagella  and  sometimes  endospores. 
Pseudomonas. — With  polar  flagella,  seldom  with  endospores. 
Spirillaceae. — Cells  forming  spirals  or  parts  of  spirals,  dividing  in  the  transverse 
plane  only. 

Spirosoma. — Inflexible  cells  without  organs  of  locomotion. 
Microspira. — Inflexible  cells  with  from  one  to  three  polar  flagella. 
Spirillum. — Inflexible  cells  with  from  five  to  twenty  curved  polar  flagella, 

forming  tufts. 
Spirochceta. — Flexible  undulating  cells  without  flagella,  but  possibly  a 

lateral  undulating  membrane. 

Chlamydobacteriaceae. — Cells    of    different   forms,    united  in    branches    or 
unbranched  chains  and  provided  with  capsules  or  envelope. 
Streptothrix. — Elongated  cells  in  non-branching  chains,  divisions  in  one 

plane  only;  reproducing  by  non-motile -conidia. 

Cladothrix. — Branching  chains,  dividing  in  one  plane  only.     Motile  repro- 
ductive cells  (Zoogonidia)  with  polar  flagella. 

4 


CLASSIFICATION  5 

Crenothrix. — Unbranched  chains,  dividing  in  three  planes,  forming  rounded 
daughter  cells. 

Phragmidiothrix. — Cells    first    unbranched,    with   very    delicate    sheath, 
dividing  in  three  planes  and  sometimes  branching  later. 

Thiothrix. — Unbranched,  non-motile  threads,  containing  sulphur  granules, 

dividing  in  one  plane  only. 

Beggiatoaceae. — Cells    without    capsules,    joined    in    unbranched    threads; 
provided  with  an  undulating  membrane. 

Beggiatoa. — Cells  with  the  characteristics  of  the  family,  containing  sulphur 
granules. 

Orla  Jensen's  classification  is  based  upon  the  nutritive  characteristics.  In 
this  the  bacteria  are  grouped  according  as  they  are:  (i)  autotrophic,  i.e.,  able 
to  form  protein  and  carbohydrates  from  inorganic  carbon  and  nitrogen;  (2) 
semi-autotrophic,  i.e.,  require  organic  carbon  compounds,  but  able  to  form 
protein  from  inorganic  nitrogen;  or  (3)  heterotrophic,  i.e.,  requiring  for  nourish- 
ment organic  compounds  of  both  carbon  and  nitrogen. 

There  are  excellent  reasons  for  accepting  the  suggestion  of  Victor  Vaughan, 
who  believes  it  expedient  to  consider  bacteria  as  such,  and  to  classify  them  as  a 
separate  group  of  living  organisms,  and  not  as  a  branch  of  either  the  vegetable 
or  animal  kingdoms. 

Some  bacteria  are  motile,  and  progress  either  by  an  undulatory  movement,  a 
screw-like  rotary  movement,  or  as  darts;  the  latter  possess  extremely  fine,  long 
thread-like  processes,  called  flagella;  these  are  believed  to  be  the  organs  of 
locomotion. 

"Structurally,  bacteria  consist  of  protoplasm,  which  at  the  periphery  is 
condensed  to  form  a  layer  resembling  a  cell  wall.  While  a  nucleus  has  not  been 
demonstrated,  it  is  probable  that  practically  the  whole  cell  is  composed  of 
nuclear  matter,  there  being  but  a  thin  peripheral  layer  of  perinuclear  proto- 
plasm" (Coplin). 

Some  bacteria  are  surrounded  by  a  capsule. 

The  chemical  composition  of  bacteria  varies  for  different  organisms  and  is 
somewhat  dependent  upon  the  nutritive  substances  upon  which  they  live. 
From  75  to  85  per  cent,  is  water,  5  to  10  per  cent,  protein,  5  to  25  per  cent,  fats 
and  i  to  2  per  cent,  ash;  chlorophyl  is  not  found. 

When  environment  becomes  adverse  to  further  development,  some  bacteria 
can  transform  into  spores.  Briefly  stated,  the  first  sign  of  endospore  formation 
is  a  granular  appearance  of  the  cell  protoplasm;  these  granules  increase  in  size, 
collect  in  one  part  of  the  cell  and  coalesce,  forming  a  round,  highly  refractile 
body,  larger  in  diameter  than  the  bacillus  and  hence  causing  a  bulging  of  the  cell 
wall.  The  cell  protoplasm  gradually,  and  in  increasing  amount,  concentrates 
in  the  endospore;  eventually  the  bacillus  has  disappeared  and  in  its  place  remains 
only  the  endospore,  globular,  highly  refractile,  containing  very  little  water,  very 
much  more  resistant  to  germicides  than  the  bacillus  from  which  it  sprung. 
Spore  formation  is  not  a  method  of  reproduction  or  propagation.  It  is  a  device 
to  resist  destruction,  a  process  of  involution. 


MEDICAL  BACTERIOLOGY 

Arthrospore  formation  has  been  described  by  some  authorities  and  denied 
by  others. 

Occasionally  a  culture  of  cocci — especially  staphylococci — is  found  which  is 
much  more  resistant  to^the  germicidal  action  of  heat,  chemicals,  etc.,  than  the 
average,  the  resistance  at  times  being  so  great  as  to  suggest  the  presence  of 
spores.  Stained  preparations  from  such  resistant  cultures  frequently  show  some 
organisms  which  differ  from  the  majority  of  their  fellows  in  size;  they  are  50 
to  100  per  cent,  larger  than  normal;  some  of  them  stain  normally  and  others 
are  resistant  to  staining.  These  bodies  are  considered  to  be  spores  (arthro- 
spores)  by  some. 

There  are  a  few  bacteria  that  thrive  at  o°C.  (psychrophilic)  and  others  that 
flourish  at  temperatures  above  7o°C.,  (thermophilic)  but  the  majority  develop 
most  luxuriantly  at  or  near  the  temperature  of  the  human  body.  Generally, 
warmth,  moisture  and  protection  from  direct  sun  rays,  are  required  for  bacterial 
development. 

Among  both  saprophytic  and  pathogenic  bacteria,  there  are  species  which 
flourish  in  an  atmosphere  containing  oxygen,  as  air,  and  die  or  change  into  spores 
when  placed  in  an  atmosphere  devoid  of  oxygen — these  are  called  obligate 
aerobic  bacteria.  There  are  obligate  anaerobic  bacteria  which  thrive  only  in 
an  atmosphere  free  from  oxygen  and  do  not  propagate  when  exposed  to  air. 

Some  bacteria  grow  best  in  air,  but  can  grow  to  a  lesser  degree  in  the  absence 
of  air  or  oxygen — these  are  called  aerobic  and  facultative  anaerobic.  There  are 
bacteria,  however,  which  are  anaerobic  and  facultative  aerobic,  and  some  that 
grow  equally  well,  whether  in  an  aerobic  or  anaerobic  environment. 

The  life  of  bacilli  is  very  variable  and  is  influenced  by  environment;  in  this 
respect  different  species  show  marked  variations. 

In  a  resting,  inactive,  or  dormant  state,  some  species  survive  for  months  or 
years.  In  full,  continuous  activity,  the  life  of  a  single  organism  is  a  matter  of 
minutes,  from  16  to  20  minutes  for  the  bacillus  coli  communis. 

According  to  A.  Fischer,  under  ideal  conditions,  1,600,000,000,000,000  ba- 
cilli would  develop  from  a  single  organism  in  24  hours.  Of  course,  ideal  con- 
ditions for  propagation  never  exist  and  increase  is  always  much  less  than  the 
potential  reproductive  power  of  any  organism. 

Just  as  for  the  individual,  there  is  a  limit  to  the  growth  and  life  of  colonies  of 
bacteria,  dependent  upon  available  food,  environment  and  an  inherent  tend- 
ency to  involute  and  die  in  the  course  of  time. 

Most  bacteria  are  saprophytic.  They  live  on  dead  animal  and  vegetable 
matter  and  by  their  activity,  rapidly  change  its  character,  splitting  it  into  simpler 
compounds,  liberating  water,  carbonic  acid,  ammonia  and  ptomains.  Sapro- 
phytic bacteria,  with  a  few  exceptions,  are  unable  to  survive  in  living  animal 
bodies  and  cannot  directly  cause  disease.  Indirectly  they  are  capable  of  harm. 
Lodged  upon  the  dead  cells  covering  the  surface  of  the  body  of  a  living  animal, 
feeding  upon  these  dead  cells  and  splitting  them  into  simpler  compounds,  some 
of  which  are  irritant  or  toxic  to  adjacent  living  cells,  they  injure  and  impair 
the  function  of  the  living  cells  and  enhance  the  possibility  of  entrance  at  that 
point  of  pathogenic  bacteria. 


CLASSIFICATION  7 

When  saprophytic  bacteria  gain  access  to  meat,  milk,  ice  cream,  canned 
vegetables,  etc.,  they  sometimes  produce  chemical  changes  which  result  in 
poisoning  when  these  articles  are  ingested. 

Parasitic  bacteria  occur  chiefly .  or  exclusively  upon  living  animal  and 
vegetable  bodies.  Some  are  entirely  beneficent,  others  are  injurious  or  de- 
structive to  certain  species  of  animals  or  vegetables. 

Pathogenic  bacteria  are  those  which  directly  cause  disease.  Many  of 
them  have  the  faculty  of  living  either  as  saprophytes  or  parasites  and  hence 
are  almost  ubiquitous,  commonly  present  in  air,  water  and  soil.  Others  are 
exclusively  parasitic.  Indeed,  the  requirements  of  some  parasitic  bacteria  are 
so  exacting  that  their  occurrence  is  limited  to  a  single  species  of  plant  or  animal 
life. 

There  are  three  groups  of  bacteria  distinguished  by  their  shape:  bacilli, 
which  are  rod-shaped,  cocci,  which  are  spherical,  and  spirilla,  curved  or  spiral- 
shaped  organisms.  Morphologically,  each  of  these  three  groups  show  numerous 
subdivisions. 

Cocci  or  micrococci  when  observed  singly  are  spherical  or  nearly  so  and  are 
nearly  equal  in  size,  regardless  of  species.  Six  forms  are  observed: 

1 .  Staphylococci  arranged  in  irregular  masses,  said  to  resemble  bunches  of 
grapes.     Frequently  the  manipulations  incident  to  placing  them  on  a  glass 
slide  and  staining  or  otherwise  preparing  for  microscopic  study,  destroys  the 
bunch  of  grape-like  arrangement  so  that  we  see  Staphylococci  singly,  in  little 
irregular  clumps  of  two,  three  or  more  elements  as  well  as  in  bunches. 

2.  Streptococci  occur  in  chains.     These  chains  may  be  long  or  short,  three, 
four,  five  or  more  cocci  in  a  row  forming  a  short  chain;  these  short  chains  are 
usually  straight.     The  longer  chains,  some  of  which  are  composed  of  50  or  more 
cocci,  may  be  straight  or  curved  or  tangled  just  like  a  piece  of  rope. 

3.  Tetrads  are  cocci  that  appear  in  groups  of  four. 

4.  Sarcina  are  cocci  arranged  in  cubes  showing  four,  eight,  twelve  or  sixteen 
elements  on  each  side  of  the  cube  observed. 

5.  Diplococci  those  arranged  in  pairs,  the  surfaces  in  apposition  being 
somewhat  flattened,  similar  to  coffee  beans. 

6.  Diplococci  arranged  in  pairs,  without  flattening  of  adjacent  surfaces, 
elements  being  either  spherical  or  lancet  shaped.     Some  of  these  latter  are 
encapsulated. 

Pathogenic  cocci  do  not  possess  the  power  of  locomotion.  Spore  formation 
by  cocci  is  a  mooted  question;  arthrospore  formation  may  be  a  property  of  some 
species. 

BACILLI 

Bacilli  as  a  class  are  rod-shaped;  some  are  so  short  and  plump  as  to  appear 
ovoid,  others  are  distinctly  rod-shaped.  They  vary  considerably  in  size.  Ob- 
serving the  smallest,  it  is  difficult  or  impossible  to  determine  whether  or  not 
their  ends  are  square  or  round,  but  this  can  be  noticed  when  observing  large 
bacilli. 

There  are  long,  wide  bacilli  having  rounded  ends,  long  wide  bacilli  with 


8  MEDICAL  BACTERIOLOGY 

square  ends,  long  slender  bacilli,  some  with  round,  others  with  square  ends, 
short  slender  and  short  plump  bacilli,  some  having  round  and  others  square 
ends;  bacilli  capable  of  locomotion  and  others  that  are  not.  Some  species  of 
bacilli,  but  not  all,  have  the  faculty  of  producing  spores  (endospores). 

SPIRILLA 

Spirilla  show  marked  variations  in  size  just  as  bacilli  do.  They  may  be 
divided  into  three  groups: 

1.  Rigid  nonmotile  spirals  (spirosoma). 

2.  Rigid  motile  spirals  (spirillum). 

3.  Flexible,  motile  spirals  (spirochaeta). 

Spirilla  propagate  by  cell  division,  cleavage  being  in  some  instances  longi- 
tudinal as  well  as  transverse.  Spore  formation  is  unknown. 

The  family  and  genus  characteristics  of  bacteria  are  permanent  so  far  as 
is  known.  Each  species  is  a  distinct  entity;  staphylococci  never  become 
gonococci,  colon  bacilli  never  become  anthrax  bacilli,  tubercle  bacilli  are  al- 
ways tubercle  bacilli,  they  never  produce  anything  but  tubercle  bacilli;  muta- 
tion from  one  form  to  another  has  not  been  proved. 

Spontaneous  development  of  bacteria  does  not  occur;  all  bacteria  origi- 
nate from  bacteria.  There  is  no  other  form  of  development. 

The  term  bacteria  is  used  to  designate  rod-shaped  schizomycetes,  it  is  also 
used  when  referring  to  the  entire  group  of  schizomycetes,  and  in  its  broadest 
sense  includes  other  higher  groups  of  organisms  conveniently  classified  as 
"higher  forms  of  bacteria." 

The  higher  bacteria  embrace  the  actinomyces,  caldothrices,  leptothrices, 
blastomycetes  and  hyphomycetes  (see  page  135). 

Bacteria  may  be  differentiated  and  classified  according  to  their  physiology, 
some  species  otherwise  indistinguishable  have  peculiar  secretions  and  others  are 
distinguished  by  the  physical  and  chemical  changes  they  produce  in  the  medium 
upon  which  they  grow. 

For  those  whose  work  frequently  necessitates  the  identification  of  bacteria 
of  soil,  water  and  foodstuffs,  "A  Manual  of  Determinative  Bacteriology" 
by  F.  D.  Chester  (The  Ma,cmillan  Co.,  New  York)  is  a  most  valuable  aid. 

It  is  highly  important  that  as  soon  as  possible  we  can  arrive  at  the  use  of  a 
proper  and  uniform  nomenclature  for  bacteria.  At  the  present  time,  unfor- 
tunately, such  is  not  in  use.  Advanced  students  are  earnestly  recommended 
to  consider  this  subject  and  to  read  especially  "  Vuillemin — Genera  Schizomy- 
cetum,  Annales  Mycologici,  xi,  512-527."  Also  Buchanan,  R.  E.,  Journal  of 
Bacteriology,  vol.  i,  No.  6,  pp.  591-596,  November,  1916. 


CHAPTER  III 
MICROSCOPY 

Bacteriology  is  an  outgrowth  of  microscopy.  For  many  years  the  study  of 
bacteria  was  confined  to  microscopic  observations.  At  present  other  methods 
described  in  the  following  chapters,  are  also  employed  but  microscopic  studies 
are  still  a  large  and  indispensable  portion  of  most  investigations. 

For  ordinary  bacteriological  work  any  of  the  microscopes  of  standard  make 
do  equally  well,  whether  of  American  or  European  manufacture.  If  but  one 
eyepiece  or  ocular  is  obtained  with  the  microscope,  a  4  or  6  will  be  found  most 
satisfactory.  At  least  three  objectives  are  required,  %  inch,  %  inch  and  ^2 
inch.  The  %-inch  should  have  a  working  distance  that  will  permit  blood-cell 
counting  and  the  J^2  inch  objective  is  an  oil  immersion  lens.  Although  a 
mechanical  stage  is  not  essential,  it  is  a  great  convenience  and  often  a  time-saving 
adjunct. 

Microscopes  are  constructed  to  rest  upon  and  be  supported  by  the  base. 
When  it  is  necessary  to  move  or  carry  one  from  place  to  place  the  safest  way  is 
to  grasp  the  instrument  below  the  stage;  never  lift  it  by  the  barrel,  coarse  ad- 
justment or  fine  adjustment.  Hold  it  upright,  do  not  tilt  it;  failure  to  observe 
these  precautions  in  handling,  often  results  in  injury  to  the  instrument. 

Dust,  acid  fumes  and  continuous  exposure  to  direct  sunlight  are  injurious; 
therefore,  when  not  in  use  a  microscope  is  protected  by  a  suitable  covering. 

Proper  illumination  is  very  important.  Direct  sunlight  is  not  desirable, 
north  light,  especially  from  a  white  cloud,  is  the  best  natural  light.  If  gas  light 
is  used  it  should  come  from  a  Welsbach  burner.  Incandescent  electric  lights 
should  have  frosted  globes  and  be  100  candlepower  or  more. 

When  unstained  preparations  are  to  be  examined,  as  in  the  observation  of 
hanging-drops,  agglutination  tests  and  red  blood-cell  counting,  oblique  illumina- 
tion will  generally  be  most  satisfactory;  when  examining  stained  bacteria  and 
tissue,  central  light  is  best. 

ILLUSTRATION  OF  MICROSCOPE  INDICATING  THE  VARIOUS  PARTS 

To  bfing  the  rays  of  light  upon  the  object  to  be  examined,  the  diaphragm 
must  be  open.  Beginners  frequently  overlook  this.  The  mirror  is  manipu- 
lated until  the  best  possible  illumination  is  obtained,  using  the  plane  side  of  the 
mirror  usually.  Then  by  focusing  the  condenser,  one  determines  at  what 
distance  from  the  object  it  gives  best  results.  Start  with  the  condenser  flush 
with  the  stage. 

When  using  a  low-power  objective,  the  %  or  J£,  one  may  look  in  the  ocular 
and  focus  downward  until  the  field  is  in  focus.  This,  however,  is  a  dangerous 
procedure  when  using  a  high-power  objective. 

Q 


10  MEDICAL  BACTERIOLOGY 


If  the  oil  immersion  lens  is  to  be  employed,  first  place  a  drop  of  immersion 
oil  on  the  slide  or  cover  glass  and  with  the  eye  fixed  on  the  objective  focus  down- 
ward until  the  objective  touches  the  oil  and  almost,  but  never  quite  touches 
the  cover  glass.  Then  look  in  the  ocular  and  slowly  focus  upward  until  the  field 
comes  into  view.  Never  touch  the  cover  glass  with  the  lens. 


FIG.  i. — MICROSCOPE  SUITABLE  FOR  GENERAL  PATHOLOGIC  AND  BACTERIOLOGIC  WORK. 
a.  Ocular  or  eye-piece,  b.  Draw-tube,  c.  Rack.  d.  Milled  head  of  pinion  moving  the 
rack;  the  rack  and  pinion  (c  and  d)  together  are  called  the  coarse  adjustment,  e.  Micro- 
scopic tube.  /.  Micrometer  screw  by  which  the  fine  adjustment  is  operated,  g.  Triple 
nose-piece  or  revolver  which  receives  the  objectives,  h;  in  the  above  instrument  there  are 
three  objectives  which  in  turn  may  be  rotated  into  the  optical  axis.  i.  Stage  on  the  upper 
surface  of  which  are  clips  for  holding  the  slide  during  examination,  j.  Iris  diaphragm  in 
substage  condenser;  the  diaphragm  permits  variation  in  the  quantity  of  light  admitted,  and 
the  condenser  properly  focuses  the  rays  on  the  object  examined,  k.  Screw  for  raising  and 
lowering  the  condenser  by  which  the  latter,  when  not  in  use,  may  be  thrown  to  the  side.  /. 
Mirror  for  reflecting  light  into  the  optical  axis  of  the  instrument,  m.  Inclination  joint  per- 
mitting inclination  of  the  instrument.  The  vertical  column  below  the  inclination  joint  is 
called  the  pillar  and  is  solidly  joined  to  the  large,  heavy,  horseshoe  base  supporting  the 
instrument. 


A  focus  is  first  obtained  with  the  coarse  adjustment;  not  until  the  best  focus 
possible  to  obtain  with  it  has  been  reached,  do  we  use  the  fine  adjustment. 

The  fine  adjustment  has  a  very  limited  range  and  should  never  be  given  more 
than  half  a  turn  one  way  or  the  other.  It  is  delicate,  easily  injured,  and  once 
out  of  order  requires  the  services  of  a  skilled  mechanic  to  adjust  it. 

When  any  part  of  the  microscope  becomes  loose  or  out  of  order,  unless  one 


MICROSCOPY  1 1 

is  experienced  in  correcting  such  faults,  it  is  best  to  return  the  instrument  to 
the  maker  for  repairs. 

Important  as  proper  illumination  and  focusing  are,  satisfactory  observations 
cannot  be  made  if  the  lenses  be  dirty,  therefore,  they  are  carefully  protected 
from  dust  and  moisture.  Keeping  an  ocular  in  the  instrument  when  not  in 
use,  prevents  dust  from  entering  the  tube  and  objective.  When  the  field  of 
vision  is  obscured  by  specks,  they  are  usually  upon  the  ocular.  To  determine 
this,  rotate  the  ocular  while  looking  through  it.  If  the  dust  is  on  the  ocular,  the 
specks  move  when  the  ocular  does.  Wipe  off  the  most  exposed  surface  first,  if 
that  does  not  correct  the  fault,  carefully  clean  all  the  surfaces  of  the  ocular  lenses, 
one  after  the  other  until  the  ocular  is  perfectly  clear.  Should  an  objective  be 
dirty,  first  clean  the  outside,  if  there  is  dust  inside  an  ocular,  try  to  remove  it 
with  a  fine  camel's  hair  brush.  Never  take  an  ocular  apart;  if  that  seems  neces- 
sary, let  the  maker  do  it.  Only  fine,  soft,  clean  linen  or  silk  or  Japanese  lens 
paper  should  be  used  to  wipe  lenses.  They  should  never  be  rubbed  hard. 
If  a  solvent  is  required  to  clean  lenses,  wet  the  lens  paper  with  xylol  or  chloro- 
form, nothing  else,  and  rapidly  dry  the  lens  after  wiping.  Immersion  oil  should 
never  be  left  on  a  lens  when  not  in  use;  it  should  be  wiped  off.  The  substage 
condenser  and  mirror  require  the  same  careful  attention  and  must  be  as  clean 
as  oculars  and  objectives.  Should  a  lens  be  scratched  or  its  finish  etched  or 
marred,  the  services  of  a  lens  grinder  will  be  required  to  restore  it. 

THE  DARK-FIELD  MICROSCOPE 

Unstained  microorganisms  are  most  distinctly  visible  and  their  motility 
and  morphology  can  be  most  accurately  observed  when  they  are  situated  in  a 
medium  that  has  a  dark  background  and.  are  illuminated  by  rays  of  light  that 
pass  obliquely  through  the  fluid  between  slide  and  cover  glass  without  entering 
the  objective.  In  fact,  organisms  such  as  the  treponema  pallidum,  which  can- 
not be  satisfactorily  observed  in  ordinary  hanging-drop  preparations,  are 
distinctly  visible  and  clearly  outlined  in  dark-field  preparations,  notwithstanding 
the  fact  that  magnification  is  the  same  in  each  case. 

For  this  desirable  method  of  observation  one  requires  a  microscope  fitted 
with  a  dark-field  condenser.  Such  a  condenser  can  be  affixed  to  any  ordinary 
microscope  but  on  account  of  the  time  necessary  to  properly  adjust  the  micro- 
scope for  this  work,  the  only  practical  method  for  routine  examinations,  at 
present,  is  to  equip  a  microscope  with  a  dark  field,  adjust  it,  and  fix  it  in  position 
and  only  use  it  for  this  method  of  observation. 

The  one  great  value  of  this  apparatus,  at  present,  in  routine  diagnostic  work, 
is  in  the  examination  of  fluids  and  scrapings  from  suspicious  lesions  to  detect 
the  presence  of  the  treponema  pallidum. 

More  intense  illumination  is  required  for  the  dark-field  microscope  than  for 
the  ordinary  and  can  be  obtained  most  satisfactorily  from  one  of  the  numerous 
electric  lamps  made  especially  for  the  purpose.  A  lens  or  round  flask  filled 
with  water  must  be  so  placed  between  the  source  of  light  and  the  microscope, 
that  by  manipulating  the  mirror  of  the  microscope  it  is  possible  to  get  the  whole 


12  MEDICAL  BACTERIOLOGY 

surface  of  it  brightly  and  uniformly  illuminated,  at  the  same  time  the  image 
of  the  source  of  light  appears  clearly  denned  on  the  mirror. 

The  dark  field  must  be  accurately  centered  by  moving  the  screws  on  the 
side  of  it,  while  looking  through  a  low-power  objective  (without  eyepiece  in 
tube)  until  the  center  of  the  field  is  brightly  illuminated  and  all  shadows  and 
haloes  disappear. 

When  proper  adjustment  has  been  achieved,  the  exact  location  of  lamp, 
condenser  and  microscope  on  the  table  should  be  noted. 

Though  not  absolutely  necessary,  it  is  much  more  satisfactory  to  make 
observations  with  this  microscope  in  a  dark  room  than  elsewhere. 

Only  slides  and  cover  glasses  of  the  thickness  for  which  the  apparatus  has 
been  constructed  should  be  used. 

PREPARATION  OF  SPECIMENS  FOR  EXAMINATION 

In  producing  fluid  or  scrapings  for  examination  for  treponema  pallidum, 
it  is  important  to  avoid  bleeding  and  to  obtain  the  specimen  free  of  blood. 
Fluid  can  be  obtained  from  macules  and  papules  by  gently  pinching  them  with 
a  hemostat  and  nicking  the  elevated  surface  with  a  sharp  knife.  If  done  care- 
fully, several  drops  of  clear  serum,  free  of  blood  cells,  can  be  expressed-  Speci- 
mens can  similarly  be  obtained  from  indurated  surfaces  and  the  margins  of 
ulcers. 

Lesions  should  not  be  washed  or  dressed  with  any  antiseptic  for  at  least 
24  hours  before  specimens  are  obtained.  About  6  hours  before  obtaining  the 
fluid  they  should  be  gently  cleansed  with  normal  salt  solution  and  covered  with 
a  gauze  dressing,  moistened  with  salt  solution,  which  dressing  is  not  disturbed 
until  the  material  for  examination  is  collected. 

From  macules,  papules  and  ulcers,  material  for  examination  can  be  collected 
and  handled  most  easily  and  safely  with  straight,  capillary  glass  tubes,  about 
6  inches  long  (Wright's  tubes).  Fluid  from  enlarged  glands  is  withdrawn  with 
a  syringe  and  needle. 

Examination  of  such  specimens  should  be  made  as  soon  as  possible — never 
more  than  an  hour — after  they  are  collected. 

A  drop  of  warm,  sterile,  salt  solution  is  placed  on  the  center  of  each  of  a 
number  of  perfectly  clean  slides,  and  a  drop  of  the  fluid  or  scrapings  to  be  ex- 
amined is  mixed  with  it.  The  mixture  should  form  a  thin  film,  free  of  air 
bubbles,  and  occupy  the  entire  space  between  the  slide  and  the  cover  glass  that 
is  dropped  upon  it. 

The  preparations  are  luted  with  melted  vaseline  or  paraffin,  a  drop  of  im- 
mersion oil  is  placed  on  the  dark-field  condenser  and  the  slide  placed  upon  it, 
another  drop  of  immersion  oil  is  placed  on  the  cover  glass  and  the  oil  immersion 
lens  brought  into  contact  with  it.  The  preparation  is  now  ready  to  examine 
when  brought  into  focus. 

SLIDES  AND  COVER  GLASSES 

Slides  and  cover  glasses  when  purchased  are  seldom,  if  ever,  clean  enough 
for  use.  The  easiest  and  most  satisfactory  method  of  cleaning  is  to  jmmerse 


MICROSCOPY  13 

in  spiritus  aetheris  nitrosi,  dry  and  polish  with  fine  linen  or  paper.  No  matter 
how  packed,  slides  and  covers  cleaned  in  bulk  for  future  work  always  collect 
some  dirt  before  they  are  required.  To  avoid  unnecessary  repetition,  slides 
and  cover  glasses  are  placed  in  jars  containing  spiritus  aetheris  nitrosi  and  dried 
and  polished  immediately  before  use. 

When  bacteria  are  to  be  examined  unstained  and  particularly  when  motility 
or  agglutination  is  to  be  observed,  the  best  method  is  to  make  a  hanging-drop 
preparation.  For  this  purpose  slides  are  made  with  a  concave  depression  in 
the  center  of  one  side.  A  drop  of  the  fluid  to  be  examined  is  placed  on  the  center 
of  a  cover  glass,  the  cover  glass  is  inverted  and  placed  over  the  cavity  of  the  slide 


FIG.  2. — HANGING-DROP  PREPARATION. 

so  that  the  drop  of  fluid  hanging  on  it  is  suspended  in  the  depression  on  the 
slide,  without  touching  the  slide  at  any  point.  To  prevent  the  slide  from  moving 
and  to  exclude  air  several  tiny  drops  of  immersion  oil  or  vaselin  are  placed  on  the 
slide  near  the  concave  depression,  before  placing  the  cover  glass  upon  it.  It  is 
important  that  the  drop  of  fluid  be  on  the  center  of  the  cover  glass,  suspended 
in  the  concave  depression  without  touching  the  slide,  and  that  the  slide  be  kept 
level,  neither  tilting  it  before  placing  on  the  stage  of  the  microscope  nor  while  it 


FIG.  3. — STEWART'S  COVER-GLASS  FORCEPS 

Method  of  holding  cover-glass  and  applying  the  stain.  Convenient  for  blood  work,  and 
absolutely  necessary  for  bacteriologic  work;  useful  also  in  sputum  examination,  etc.  The 
lower  instrument  shows  a  cover-glass  with  stain  in  position.  If  the  cover-glass  be  grasped 
with  the  film  or  spread  side  toward  the  side  of  the  forceps  that  has  the  circular  bend  intended 
to  fit  the  thumb,  shown  at  A,  there  will  be  no  danger  of  losing  trace  of  the  side  containing  the 
spread,  as  this  side  of  the  forceps  is  practically  always  upward. 

is  there.  Oblique  and  not  too  bright  illumination  is  best  for  the  study  of  hang- 
ing-drops. 

Bacteria  are  most  commonly  examined  after  staining  and  must  first  be  fixed  to 
the  slide  or  cover  glass  to  prevent  subsequent  washing  from  carrying  them  away. 

If  it  is  fluid  one  wishes  to  examine,  as  when  the  bacteria  are  in  bouillon, 
milk,  etc.,  a  small  drop  is  placed  on  a  perfectly  clean  cover  glass  (which  has  been 
flamed  and  allowed  to  cool)  and  spread  in  a  thin  even  film  with  a  platinum  loop. 
Sputum,  pus  and  similar  viscid  substances  are  more  readily  spread  in  a  thin 
even  film  by  placing  a  drop  on  the  center  of  a  slide  or  cover  glass  then  dropping 
a  second  slide  or  cover  glass  on  top  of  it  and  allowing  the  material  to  spread  out 
between  them,  then  slide  the  glasses  apart. 

When  dried  substances  and  bacteria  removed  from  solid  culture  media  are 
to  be  examined,  a  drop  of  sterile  distilled  water  is  placed  on  a  slide  or  cover  glass, 


14  MEDICAL  BACTERIOLOGY 

the  dried  matter  or  bacteria  picked  up  with  a  sterile  platinum  loop  and  the  loop 
lightly  touched  to  the  drop  of  water  until  a  slight  cloudiness  appears.  The 
excess  is  then  burned  off  the  loop  and  when  it  is  cool,  the  bacteria  are  mixed  with 
the  drop  of  water  and  it  is  spread  out  in  a  thin  film  with  the  loop. 

When  the  matter  to  be  examined  has  been  spread  on  the  slide  or  cover  glass, 
whether  fluid  as  bouillon,  viscid  as  sputum,  or  solid  mixed  with  water,  it  is  fixed 
by  drying.  This  is  commonly  done  by  gently  heating  over  a  flame;  should  the 
slides  be  placed  nearer  the  flame  than  one  can  hold  one's  hand  with  comfort, 
the  organisms  may  be  destroyed  or  so  altered  as  to  become  invisible.  Some 
experienced  observers  state  that  of  two  sets  of  slides  smeared  from  one  sample 
of  sputum,  one  set  dried  or  fixed  by  gently  heating  over  a  flame,  the  other  set 
fixed  by  simply  permitting  them  to  stand  at  room  temperature  until  dry,  often 
those  fixed  without  heating  will  show  tubercle  bacilli  when  those  fixed  by  flame 
do  not.  Some  workers  prefer  fixing  their  preparations  by  placing  them  in  a 
thermostat  until  dry. 

Whether  smears  shall  be  made  upon  cover  glasses  or  slides  is  a  matter  of 
personal  choice,  determined  by  convenience.  Slides  are  less  fragile  and  easier 
to  handle  than  cover  glasses  while  making  the  smears;  on  the  other  hand,  cover 
glasses  are  easier  to  handle  when  staining. 

If  smears  are  made  on  slides,  when  fixed,  stained  and  dried  they  can  be  ex- 
amined without  mounting  a  cover  glass;  cover  glasses  when  ready  for  examina- 
tion must,  of  course,  be  mounted  on  slides. 

The  essential  points  in  making  smears  for  microscopic  examination  are  as 
follows : 

1.  Use  absolutely  clean  slides  and  covers. 

2.  Spread  the  material  in  a  thin,  even,  transparent  film. 

3.  Protect  it  from  contamination  with  dust,  etc. 

4.  Guard  against  overheating  during  fixation. 

Cover  glasses  are  usually  attached  to  slides  with  Canada  balsam  which 
should  be  free  of  acid.  A  drop  of  balsam  is  placed  on  the  slide  and  the  cover 
glass  placed  upon  it.  The  balsam  should  be  free  from  air  bubbles  and  dirt.  If 
it  has  the  proper  consistency  it  will  spread  out  in  a  thin  film.  There  should  be 
no  appreciable  elevation  of  the  cover  above  the  slide.  When  balsam  becomes  so 
thick  that  it  will  not  spread  unless  pressure  is  used,  it  should  be  thinned  out 
by  adding  to  it  xylol. 

Slides  can  be  used  repeatedly,  if  thoroughly  cleaned.  A  satisfactory  method 
is  as  follows: 

Keep  a  jar  containing  a  piece  of  toilet  soap  and  water  on  the  microscope 
table;  when  through  with  slides,  place  them  in  jar.  After  48  hours  wipe  them 
clean  and  place  in  running  water  for  Y±  hour,  wipe  dry  and  store  in  a  jar  con- 
taining spiritus  aetheris  nitrosi. 

After  a  time  slides  that  have  been  used  and  cleaned  repeatedly  become 
scratched;  scratched  slides  should  never  be  used  when  differential  staining  is  to 
be  done. 

In  a  general  laboratory  new  slides  should  be  used  for  bacteriological  work; 
afterward  they  should  be  set  aside  for  tissue  and  other  work. 


CHAPTER  IV 
STAINING 

Organisms  so  small  that  they  are  almost  or  entirely  invisible  before  staining, 
become  distinctly  visible  when  stained.  Details  unnoticeable  in  unstained 
bacteria  are  apparent  in  the  stained.  By  staining  we  are  enabled  to  distinguish 
some  species  from  others  because  of  differences  in  their  ability  to  absorb  and 
retain  certain  stains,  and  to  resist  the  decolorizing  action  of  acids  and  alcohol. 

Aqueous  solutions  of  anilin  dyes  are  most  commonly  employed  in  staining 
bacteria.  Stains  and  methods  of  staining  are  numerous.  Fortunately,  how- 
ever, there  are  about  a  half  dozen  stains  and  methods  of  staining  which  suffice 
for  all  ordinary  bacteriological  examinations. 

The  term  cubic  centimeter  (cc.)  as  used  in  this  volume  is  synonymous  with 
the  term  mil.  The  United  States  Pharmacopeia  IX  has  brought  about  a  change 
from  the  former  to  the  latter,  this  being  an  abbreviation  for  the  term  milliliter 
(the  first  three  letters  of  which  are  mil).  The  United  States  Bureau  of  Stand- 
ards regards  the  term  cc.  as  a  misnomer,  there  being  a  slight  difference  between 
the  thousandth  part  of  a  liter  and  the  cc.  In  addition  the  change  was  brought 
about  to  promote  international  conformity. 

As  this  volume  will  be  used  by  students  and  practitioners  familiar  with  the 
term  cc.,  and  not  so  familiar  with  the  better  term,  mil.,  it  has  been  thought 
advisable  to  still  retain  cc.  in  this  edition. 

LOEFFLER'S  METHYLENE  BLUE 

i  :io,ooo  (Hoo  Per  cent.)  aqueous  sol.  of  potassium  hydrate,  10  cc. 
Saturated  alcoholic  solution  of  methylene  blue,  3  cc. 

Loeffler's  methylene  blue  is  a  very  satisfactory  stain  for  most  bacteria.  The 
method  of  staining  is  as  follows: 

1.  Cover  the  preparation  to  be  stained  with  Loeffler's  methylene  blue,  allow 
the  stain  to  remain  on  the  specimen  for  i  to  5  minutes. 

2.  Wash  the  stain  off  with  water,  and  allow  the  slide  or  cover  glass  to  dry. 

3.  Mount. 

4.  Examine  specimen. 

FUCHSIN 

Saturated  alcoholic  solution  of  basic  fuchsin,  5  cc. 

Distilled  water,  95  cc. 

Apply  this  stain  in  the  same  manner  as  Loeffler's  methylene  blue. 


1 6  MEDICAL  BACTERIOLOGY 

GENTIAN  VIOLET 

Saturated  alcoholic  solution  of  gentian  violet,  5  cc. 

Distilled  water,  95  cc. 

Apply  the  same  as  Loeffler's  methylene  blue. 

EOSIN 

Eosin,  J4  0  to  y±  Gm. 
Distilled  water,  100  cc. 

Allow  stain  to  remain  on  the  specimen  for  i  minute,  then  wash  off  with  water. 
Gram's  method  of  staining  permits  the  differentiation  of  organisms  otherwise 
indistinguishable  under  the  microscope,  and  is,  therefore,  of  great  service. 

GRAM'S  METHOD 

1.  Stain  specimen  with  anilin  gentian  violet  for  3  to  5  minutes. 

2.  Wash  with  water  to  remove  excess  stain. 

3.  Apply  Gram's  solution  for  2  minutes. 

4.  Wash  specimen  with  95  per  cent,  alcohol  until  specimen  gives  off  no  more 
color. 

5.  Dry  and  examine. 

Gram  positive  bacteria  (Gram  +)  remain  a  deep  violet,  almost  black. 

Gram  negative  bacteria  (Gram  — )  are  colorless. 

When  the  anilin  gentian  violet  is  applied  to  a  slide  or  cover  glass,  all  the 
bacteria  on  it  are  stained  violet.  When  the  alcohol  is  applied  it  does  not  affect 
the  Gram  positive  bacteria,  they  remain  violet,  but  the  alcohol  takes  the  violet 
stain  out  of  the  Gram  negative  bacteria,  or  in  other  words,  decolorizes  them. 

As  unstained  bacteria  are  almost  indiscernible,  a  counter  stain  is  usually 
applied  after  staining  by  Gram's  method. 

GRAM  POSITIVE  GRAM  NEGATIVE 

Cocci 


Micro- 
cocci 
of 

Suppur- 
ation 


Staphylococcus  pycgenes  albus 

Staphylococcus  pyogenes  aureus 

Staphylococcus  pyogenes  citreus 

Micrococcus  pyogenes  fcetidus  Gonococcus 

Micrococcus  cereus  albus  Meningococcus 

Micrococcus  cereus  flavus  Micrococcus  catarrhalis 

Micrococcus  epidermidis  albus  Micrococcus  melitensis 

Streptococci 

Pneumococci 

Sarcina  lutea 

Sarcina  alba 

Sarcina  aurantiaca 
Micrococcus  tetragenus 


STAINING 


GRAM  POSITIVE  GRAM  NEGATIVE 

BACILLI  (non-spore-bearing) 


f    Diphtheria  bacillus 
Corynebacteria  ^    Pseudodiphth. 'bacilli 

[  Xerosis  bacillus 
Tubercle  bacillus 
Bacillus  leprae 


Bacillus  of  Friedlander 

Bacillus  of  influenza 

Bacillus  of  pertussis  (Bordet-Gengou) 

Bacillus  of  Morax  and  Axenfeld 

Kock-Weeks  bacillus 

Bacillus  pestis. 

Typhoid  bacillus  (Eberth) 

Paratyphoid  bacilli 

Bacillus  coli 

Bacillus  icteroides 

Bacillus  of  dysentery  (Shiga's)  and  allied  or- 
ganisms 

Bacillus  proteus  vulgaris 

and  other  members  of  the  proteus  group 

Bacillus  pyocyaneus 

Bacillus  prodigiosus 

Bacillus  mallei  (glanders) 

Bacillus  of  soft  chancre  (Ducrey's) 


BACILLI  (spore-bearing) 


Bacillus  of  tetanus 
Bacillus  aerogenes  capsulatus 
Bacillus  anthracis 
Bacillus  botulinus 
Bacillus  subtilis 


Bacillus  of  malignant  edema 
Bacillus  of  symptomatic  anthrax 


SPIRILLA 


Spirillum  and  bacillus  of  Vincent 
Spirillum  of  Asiatic  cholera 
Spirillum  and  allied  organisms 
Spirochseta  of  Obermeier 
Treponema  pertenuis 
Treponema  pallidum 


HIGHER  BACTERIA 


Leptothrices 

Cladothrices 

Streptothrices 

Saccharomycetes  (blastomycetes) 

Hyphomycetes  (mucorini) 


1 8  MEDICAL  BACTERIOLOGY 

ANILIN  WATER 

Anilin  water  is  prepared  by  adding  anilin  oil  to  distilled  water,  shaking  after 
the  addition  of  each  drop.  The  oil  is  added  until  the  water  is  saturated.  This 
water  must  be  filtered  until  entirely  free  from  visible  globules  of  oil,  even  though 
several  passages  through  filter-paper  be  necessary. 

ANILIN  GENTIAN  VIOLET 

Anilin  water,  84  c.c. 

Saturated  alcoholic  solution  of  gentian  violet,  16  cc. 

Anilin  gentian  violet  rapidly  deteriorates  and  in  a  few  days  or  weeks  is 
useless. 

GRAM'S  SOLUTION 

Iodine,  i  Gm. 

Potassium  iodide,  2  Gm. 

Distilled  water,  3  oo  cc. 

In  routine  work  Loeffler's  methylene  blue  is  generally  used  and  found  most 
satisfactory  for  staining  the  diphtheria  bacillus,  but  one  of  the  characteristics 
of  this  organism,  irregular  staining,  is  best  brought  out  by  Neisser's  method 
of  staining. 

When  stained  with  Loeffler's  methylene  blue,  one,  two  or  more  portions 
of  the  diphtheria  bacillus  stain  de  eply,  the  rest  of  the  organism  stains  faintly  or 
not  at  all,  thus  giving  a  deceptive  impression  of  its  contour.  With  the  two 
extremities  stained  deeply  and  the  intermediate  portion  stained  faintly  or  not 
at  all,  so-called  polar  staining,  or  with  both  poles  and  the  center  stained  deeply 
and  the  portions  between  the  center  and  poles  unstained,  so-called  barred  stain- 
ing or  beaded  forms,  the  organism  may  appear  more  like  several  cocci  in  a  row 
than  a  bacillus,  which  it  really  is. 

By  Neisser's  method  the  entire  bacillus  is  stained,  the  ends  or  the  ends  and 
center  blue,  intermediate  portions  light  brown.  Two  solutions  are  used  to 
obtain  this  effect,  as  follows: 

NEISSER'S  METHOD 

1.  Apply  Neisser's  acid  methylene  blue  to  specimen  for  2  to  5  minutes. 

2.  Wash  with  water  to  remove  excess  stain. 

3.  Apply  Bismarck  brown  for  i  minute. 

4.  Wash  with  water. 

5.  Dry,  mount  and  examine. 

NEISSER'S  ACID  METHYLENE  BLUE 

1.  Methylene  blue,  o.i  Gm.  3.  Glacial  acetic  acid,  5  cc. 

2.  Alcohol,  2  cc.  4.  Distilled  water,  95  cc. 

.  First  mix  the  methylene  blue  and  alcohol  together,  then  mix  the  glacial 
acetic  acid  and  distilled  water  together,  finally  add  the  acid  solution  to  the 
alcoholic  solution,  and  filter. 


STAINING  I Q 

BISMARCK  BROWN 

Bismarck  brown,  0.2  Gm. 

Boiling  distilled  water,  100  cc. 

Mix  together  and  filter. 

Most  bacteria  when  stained  can  be  decolorized  by  washing  with  acids. 
But  there  are  some  bacteria  that  cannot  be  decolorized  by  acids  after  they  have 
been  stained.  These  we  call  acid-fast.  A  number  of  organisms  which  are 
acid-fast  can  be  decolorized  with  alcohol.  They  are  acid-fast,  but  not  alcohol- 
fast.  Of  the  pathogenic  organisms  there  are  but  two  which  are  both  acid-fast 
and  alcohol-fast,  the  tubercle  bacillus  and  the  leprosy  bacillus. 

It  has  been  recently  stated  that  there  are  non-acid  fast  and  non-alcohol- 
fast  strains  of  the  leprosy  bacillus.  Practically,  the  tubercle  bacillus  is  always 
acid-  and  alcohol-fast;  but  the  degree  of  resistance  to  acid  and  alcohol  of  some 
other  organisms  at  times  simulates  that  of  the  tubercle  bacillus,  so  that  rare 
occasions  arise  when  differentiation  can  only  be  determined  by  cultural  test. 

To  demonstrate  acid-fast  bacilli,  apply  carbol  fuchsin  for  15  minutes,  wash 
with  25  per  cent,  aqueous  solution  sulphuric  acid  until  specimen  gives  off  no 
more  color,  wash  with  water,  apply  LoefHer's  methylene  blue  for  3  to  5  minutes, 
wash  in  water,  dry  and  examine. 

When  the  carbol  fuchsin  is  applied  everything  is  stained  red.  The  acid 
removes  the  stain  from  all  but  the  acid-fast  organisms,  these  remain  red,  the 
others  are  decolorized.  When  the  methylene  blue  is  added  it  does  not  affect 
the  acid-fast  organisms,  they  are  already  saturated  with  fuchsin  and  remain 
red,  but  it  tints  everything  else  blue,  so  that  the  acid-fast  organisms  appear  red 
and  the  non-acid-fast  organisms  blue. 

Bacteria  that  are  decolorized  by  acids  are  also  decolorized  by  alcohol. 

To  distinguish  bacteria  that  are  acid-fast  but  not  alcohol-fast,  from  those 
which  are  both  acid-  and  alcohol-fast,  make  several  slides,  stain  some  with 
carbol  fuchsin  5  to  15  minutes,  apply  25  per  cent,  aqueous  solution  of  sulphuric 
acid  for  2  or  3  minutes,  wash  with  water  and  apply  LoefBer's  methylene  blue. 
Acid-fast  organisms  will  appear  red.  The  remaining  slides  stain  with  carbol 
fuchsin  as  before,  then  apply  either  absolute  alcohol  or  5  per  cent,  alcoholic 
solution  of  sulphuric  acid  or  3  per  cent,  alcoholic  solution  of  nitric  acid  for  10 
to  15  minutes.  Wash  with  water  and  counter  stain  with  LoefHer's  methylene 
blue.  Bacteria  that  are  both  acid-fast  and  alcohol-fast  will  appear  red,  those 
which  are  acid-fast  but  not  alcohol-fast  will  appear  blue. 

In  'diagnostic  work,  when  examining  sputum,  etc.,  for  tubercle  bacilli,  time 
and  labor  are  commonly  saved  by  combining  counter  stain  and  decolorizing 
agent,  as  in  Gabbet's  solution  and  Pappenheim's  solution,  which  are  the  ones 
commonly  used.  After  staining  with  carbol  fuchsin  5  to  15  minutes,  Pappen- 
heim's solution  is  applied  for  15  minutes,  then  wash  in  water,  dry  and  mount. 
Organisms  which  are  both  acid-  and  alcohol-fast,  appear  red,  the  others  blue. 

When  Gabbet's  solution  is  used  it  is  applied  for  2  minutes,  after  staining 
with  carbol  fuchsin.  The  specimen  is  then  washed  with  water,  dried  and  ex- 


20  MEDICAL  BACTERIOLOGY 


amined.     Those  organisms  which  are  only  acid-fast,  as  well  as  those  which  are 
acid-fast  and  alcohol-fast,  appear  red,  other  bacteria  blue. 


CARBOL  FUCHSIN 


Saturated  alcoholic  solution  of  fuchsin,  i  cc. 

Five  per  cent,  aqueous  solution  of  carbolic  acid,  9.  cc. 

PAPPENHEIM'S  SOLUTION 

Coralin  (rosalic  acid),  i  Gm. 
Absolute  alcohol,  100  cc. 

Mix  acid  and  alcohol  together;  add  methylene  blue  to  saturation  and  20  cc. 
of  glycerin. 

GABBET'S  SOLUTION 

Methylene  blue,  2  Gm. 

25  per  cent,  aqueous  solution  sulphuric  acid,  100  cc. 

The  flagella  possessed  by  motile  bacilli  cannot  be  demonstrated  by  any  of 
the  methods  of  staining  previously  described.  Many  stains  have  been  devised 
especially  for  this  purpose;  McCrorie's  is  probably  the  best. 

McCRORIE'S  FLAGELLA  STAIN 

Saturated  alcoholic  solution  of  night  blue,  i  cc. 

10  per  cent,  aqueous  solution  of  tannic  acid,  i  cc. 

10  per  cent,  aqueous  solution  of  alum,  i  cc. 

Eighteen-hour-old  agar  cultures  are  used.  Thin  spreads  are  carefully  dried 
at  a  temperature  not  greater  than  5o°C.  Apply  stain  for  2  minutes,  then  gradu- 
ally heat  until  steam  rises,  wash,  dry  and  examine. 

The  demonstration  of  flagella  is  difficult  and  of  no  practical  importance. 

To.  stain  the  capsule  with  which  some  organisms  are  surrounded  requires 
special  stains  and  Muir's  method  will  be  found  satisfactory.  Only  very  thin 
films  should  be  made  to  obtain  capsule  staining. 

MUIR'S  METHOD 

1.  Stain  with  carbol  fuchsin  for  30  seconds,  and  steam. 

2.  Wash  with  95  per  cent,  alcohol  for  several  seconds. 

3.  Wash  with  water. 

4.  Apply  mordant  for  5  to  10  seconds. 

5.  Wash  in  water. 

6.  Wash  with  alcohol  for  i  minute. 

7.  Wash  in  water. 

8.  Counter  stain  with  methylene  blue  for  J^  minute. 

9.  Dehydrate  in  alcohol. 

10.  Clear  in  xylol  and  mount. 


STAINING  2 1 

MORDANT  FOR  CAPSULE  STAINING  (MUIR'S  METHOD) 

Saturated  aqueous  solution  mercuric  chloride,  2  cc. 

25  per  cent,  aqueous  solution  tannic  acid,  2  cc.  * 

Saturated  aqueous  solution  potash  alum,  5  cc. 

Spores  do  not  stain  by  the  methods  generally  employed  for  bacteria. 

When  a  bacillus  containing  an  endospore  is  stained  with  eosin,  fuchsin  or 
methylene  blue,  the  bacillus  red  or  blue,  the  spore  colorless,  by  contrast  it  is 
apparent. 

To  stain  the  spores  and  maintain  the  contrast,  use  the  following  method: 

METHOD  FOR  STAINING  SPORES 

1.  Stain  with  carbol  fuchsin  for  15  minutes. 

2.  Wash  with  i  per  cent,  aqueous  solution  of  sulphuric  acid  until  bleaching 
is  noticed. 

3.  Wash  in  water. 

4.  Apply  methylene  blue  for  30  seconds. 

5.  Wash  in  water,  dry  and  examine. 
Spores  will  be  red  and  bacteria  blue. 

BLOOD-CELL  STAINING 

It  is  occasionally  necessary  to  stain  blood,  serous  fluids  or  exudates  con- 
taining bacteria  by  a  method  that  will  show  and  differentiate  the  white  blood 
cells.  For  this  purpose  the  preparations  are  made  and  stained  by  the  methods 
commonly  used  in  the  examination  of  blood  cells : 

A  drop  of  the  blood  or  serous  fluid  is  placed  on  the  center  of  a  square  cover 
glass  and  a  second  cover  glass  dropped  diagonally  on  it.  As  soon  as  the  fluid 
ceases  to  spread  the  glasses  are  slid  apart;  or,  a  drop  of  the  fluid  is  placed  on  a 
slide,  near  one  end,  and  is  drawn  along  the  slide  with  the  edge  of  a  second  slide 
held  at  an  angle  of  about  45°.  By  whichever  method  the  spread  is  made,  it 
must  be  done  rapidly  and  the  film  must  be  thin  and  even.  It  is  gently  heated 
until  dry  and  then  stained  with  Romanowsky's,  Giemsa's,  or  any  of  the  modifica- 
tions of  these.  The  preparation  is  covered  with  the  stain,  from  i  to  2  minutes 
later  distilled  water  is  added  to  the  stain  until  a  metallic  scum  appears,  usually 
it  requires  as  much  water  as  stain  to  effect  this:  5  to  10  minutes  later  the  slide 
or  cover  glass  is  washed  with  water,  dried  and  examined. 

Saturated  alcoholic  solutions  of  fuchsin,  methylene  blue  and  gentian  violet 
keep  until  used.  It  is  a  great  convenience  to  have  such  solutions  in  stock. 
They  should  be  stored  in  dark  glass-stoppered  bottles.  Absolute  alcohol  is 
used  in  their  preparation  and  the  addition  of  10  Gm.  of  powdered  stain  to  each 
100  cc.  of  alcohol  is  more  than  sufficient  to  saturate  it.  When  made  saturated, 
solutions  are  thoroughly  shaken  and  then  allowed  to  sediment  for  at  least 
24  hours  before  use.  Care  must  afterwards  be  taken  when  pouring  that  no 
undissolved  particles  get  into  staining  fluids.  These  latter  must  all  be  per- 
fectly clear  and  free  of  undissolved  particles.  When  procuring  stains  it  is 
best  to  specify  "Grubler's." 


22  MEDICAL  BACTERIOLOGY 

GIEMSA'S  STAIN 

Azur  II-eosin 3.0  Gm. 

,    Azur  II o.SGm. 

Glycerin  (C.P.) 250.0  cc. 

Methyl  alcohol  (C.P.) 250 .  o  cc. 

Grind  up  dyes  in  alcohol,  then  add  glycerin.  Fix  film  in  methyl  alcohol, 
stain  5  minutes  with: 

Giemsa 14  gtt. 

H2O 10  gtt 

For  the  principles  and  mechanism  of  staining  of  microorganisms,  consult 
i'The  Microtomist's  Vade-Mecum,"  A.  B.  Lee,  P.  Blakiston's  Son  &  Co., 
Philadelphia. 


CHAPTER  V 
STERILIZATION 

Sterilization  is  the  removal  of  germs  from  matter  or  destruction  of  germs 
upon  or  in  matter.  It  can  be  accomplished  in  the  case  of  fluids,  by  nitration, 
the  addition  of  germicidal  chemicals  or  by  heat.  Solids  can  be  sterilized  by 
the  addition  of  germicidal  chemicals  or  by  heat. 

Filtration  can  sterilize  under  the  following  conditions: 

1.  When  the  pores  of  the  filter  are  too  small  to  permit  the  passage  of  bacteria. 

2.  When  the  filtrate,  uncontaminated,  flows  into  a  sterile  container. 

3.  When  the  construction  of  the  filter  and  the  connection  of  filter  to  filtrate 
container  are  such  as  to  prevent  unfiltered  fluid  and  air  from  mingling  with  the 
filtrate. 

4.  When  the  filtrate  is  sealed  in  its  container  before  exposure  to  contamina- 
tion with  germs. 

To  secure  sterilization  by  this  method  requires  the  employment  Ox  a  properly 
selected,  tightly  adjusted  and  nicely  manipulated  filter  and  filtrate  container. 
Not  easy  for  a  tyro  to  do. 

In  routine  laboratory  work,  this  method  is  seldom  employed  except  in  the 
sterilization  of  therapeutic  sera  and  such  solutions  as  would  be  injured  by  heat. 
The  filters  used  are  unglazed  porcelain  tubes  which  are  first  tested  by  passing 
through  them,  fluids  known  to  contain  bacteria  and  then  testing  the  filtrate  for 
sterility. 

Chemical  Sterilization  commonly  referred  to  as  disinfection,  is  accom- 
plished by  submerging  utensils  in  a  germicidal  solution,  or  by  mixing  germicidal 
solutions  with  fluid  and  solid  substances. 

The  efficacy  of  this  method  depends  upon  the  following: 

1.  The  selection  of  a  chemical  which  can  kill  germs. 

2.  The  selection  of  a  chemical  which  is  not  changed  to  an  inert  substance, 
without  germicidal  power,  when  brought  into  contact  with  .the  matter  con- 
taminated with  germs. 

3.  The  use  of  sufficient  quantity  of  germicide  to  destroy  all  germs. 

4.  Direct  contact  of  germicidal  agent  with  every  germ. 

5.  Maintenance  of  the  contact  for  a  sufficient  length  of  time. 

6.  The  rate  at  which  chemical  sterilization  progresses  is  in  proportion  to 
the  hydrogen  ion  concentration,  and  to  some  extent,  increased  by  the  presence 
of  sodium  chloride. 

Chemical  sterilization  is  restricted  by  these  conditions  which  must  be  ful- 
filled. It  is  further  restricted  by  the  uncertainty  of  results.  Its  employment 
is  limited  because  most  germicidal  chemicals  cannot  be  easily  removed  from 
substances  with  which  they  have  been  mixed,  therefore,  although  sterilized, 

23 


24  MEDICAL  BACTERIOLOGY 

the  substance  still  remains  unfit,  if  intended  for  human  consumption,  thera- 
peutic administration,  or  for  use  as  culture  medium,  upon  which  to  grow  bacteria. 

Chemical  sterilization  is  principally  employed  in  the  treatment  of  refuse 
matter  which  may  contain  pathogenic  organisms.  Chloroform  and  ether, 
which  can  be  removed  by  a  slight  degree  of  heating,  are  occasionally  used  for 
chemical  sterilization,  as  in  the  sterilization  of  serum. 

The  rate  at  which  chemical  sterilization  progresses  is  in  proportion  to  the 
hydrogen  ion  concentration.  This  consideration  will  explain  the  greater 
activity  shown  by  chemicals  in  aqueous  solutions  than  in  alcoholic  or  ethereal 
admixture,  since  it  has  been  shown  that  ionization  is  more  pronounced  and 
greater  in  water  than  in  other  solutions.  Sodium  chloride  and  other  chemical 
salts  when  added  to  the  solution  of  an  inorganic  disinfectant  seem  to  retard  the 
action  of  the  latter,  inasmuch  as  these  salts  tend  to  decrease  the  concentration 
of  free  ions.  However,  their  presence  with  the  organic  disinfectants,  notably 
the  phenols  and  coal-tar  derivatives,  tends  to  increase  the  bactericidal  powers 
of  such  preparations.  This  fact  is  sufficient  proof  that  the  phenols  and  their 
allied  members  act  as  an  entire  molecule  and  not  as  individual  ions. 

DISINFECTANTS  USED  IN  SOLUTION 

How  disinfectants  effect  the  injury  of  bacteria  is  a  problem  still  in  doubt. 
Some  seem  to  act  by  their  readiness  in  coagulating  the  cell  protoplasm.  Others 
act  by  their  power  of  rapidly  oxidizing  the  bacterial  body.  Some  may  produce 
injury  to  the  cell  by  their  property  of  withdrawing  water  from  its  tissues,  while 
still  others  exert  their  toxic  effect  by  penetrating  the  cell  wall  and  inactivating 
the  cell  protoplasm. 

ACIDS  AND  ALKALIES 

The  strong  inorganic  acids  and  alkalies  are  active  germicidal  agents,  due  to 
the  fact  that  they  readily  ionize.  The  acids  possess  this  property  to  a  greater 
extent  than  the  latter. 

Bichloride  of  mercury  is  one  of  the  most  extensively  used  antiseptics. 
Although  highly  efficient  under  certain  conditions,  it  is  so  readily  destroyed  and 
its  efficiency  lowered  as  commonly  used  that  it  becomes  an  undependable  dis- 
infectant. It  is  poisonous,  irritating  and  highly  toxic.  It  is  precipitated  by 
soap  and  its  precipitating  properties  by  albumen  limits  its  use  as  a  penetrating 
agent. 

Peroxide  of  hydrogen  has  been  used  extensively,  because  of  its  oxidizing 
properties.  Regarded  as  very  efficient  by  some,  it  is  considered  worthless  by 
others.  This  is  because  of  its  rapid  destruction  in  the  presence  of  organic 
matter  (especially  blood,  pus,  etc.)?  so  that  no  great  dependence  can  be  placed 
upon  its  disinfectant  properties,  unless  used  in  excessive  amounts. 

Permanganate  and  dichromate  of  potash  are  two  inorganic  salts,  which  as 
oxidizing  agents,  are  powerful  disinfectants.  These  are  also  rapidly  reduced 
and  inactivated,  as  far  as  their  bactericidal  properties  are  concerned,  by  organic 
matter. 


STERILIZATION  25 

Silver  salts. — The  silver  salts,  as  silver  nitrate,  argyrol  and  protargyrol,  are 
of  a  special  value  as  disinfectants  for  some  of  the  cocci.  This  fact  explains  the 
extensive  use  of  these  chemicals  in  the  treatment  of  gonorrheal  ophthalmia 
and  certain  types  of  sore  throat. 

The  Halogens. — Perhaps  more  commonly  used  than  any  of  the  foregoing 
inorganic  disinfectants  are  the  free  halogens,  notably  iodine  and  chlorine,  and 
to  a  minor  extent  free  bromine. 

Iodine,  both  as  terchloride  of  iodine  (IC13)  and  free  iodine  are  extremely 
strong  disinfectants.  Tincture  iodine  (U.S.P.)  or  10  per  cent,  is  a  simple  and 
most  efficient  method  of  sterilizing  the  skin. 

Chlorine,  both  free  and  available,  is  perhaps  one  of  the  most  common  of 
inorganic  germicides. 

Chloride  of  lime,  or  the  so-called  "bleaching  powder,"  is  a  substance, 
possessing  probably  the  formula  CaOCl2.  The  action  of  dilute  acids  or  air  (in 
which  we  find  COz  and  moisture,  thus  forming  carbonic  acid)  upon  this  substance 
results  in  the  liberation  of  chlorine,  so  termed  "available."  The  latter,  because 
of  its  readiness  in  destroying  vegetative  forms  of  bacteria,  when  even  in  dilute 
solutions,  has  brought  about  the  extensive  use  of  this  chemical  in  the  disinfec- 
tion of  sewage  and  water  supplies. 

.  A  preparation  which  has  been  known  for  years,  but  recently  exploited  and 
used  more  extensively,  is  the  Dakin-Carrel  solution.  This  product  is  made  like 
Labarraque's  solution  of  the  United  States  Pharmacopeia,  but  possessing  special 
precautionary  differences,  as  may  be  observed  below,  where  follow  its  details 
and  mode  of  preparation.  As  this  solution  liberates  its  chlorine  when  in  contact 
with  wounds  and  retains  its  antiseptic  properties  for  a  long  time,  it  will  be  valu- 
able only  when  it  is  in  no  way  harmful  to  the  cells  of  the  surrounding  tissues. 
This  can  be  taken  care  of  by  observing  that  the  concentration  of  available  chlo- 
rine be  uniform  and  of  the  standardized  strength,  and  that  the  product  be  free 
from  irritating  substances,  as  free  alkali,  boric  acid,  etc.  This  preparation, 
when  used  in  surgery,  is  employed  by  immersing  a]l  the  wound  surfaces 
through  a  constant  flow,  maintained  by  a  hydraulic  system.  At  the  same 
time,  all  portions  of  the  skin  surrounding  the  wound  are  protected  from  ex- 
posure to  the  chlorin  solution,  by  having  applied  to  it  sterile  gauze  strips 
with  vaseline. 

PREPARATION  OF  DAKIN'S  SOLUTION  (DAUFRESNE'S  TECHNIQUE) 

Original  letter  by  Dr.  Carrel 
(Journal  of  the  American  Medical  Association,  Dec.  9,  1916,  p.  1777) 

"Dakin's  solution  is  a  solution  of  sodium  hypochlorite  for  surgical  use,  the 
characteristics  of  which,  established  after  numerous  tests  and  a  long  practical 
experience,  are  as  follows: 

(a)  "Complete  Absence  of  Caustic  Alkali. — The  absolute  necessity  for  em- 
ploying in  the  treatment  of  wounds  a  solution  free  from  alkali  hydroxide  excludes 


26  MEDICAL  BACTERIOLOGY 

the  commercial  Javelle  water,  Labarraque's  solution  and  all  the  solutions  pre- 
pared by  any  other  procedure  than  the  following: 

(b)  "Concentration. — The  concentration  of  sodium  hypochlorite  must  be 
exactly  between  0.45  and  0.50  per  cent.  Below  0.45  per  cent,  of  hypochlorite 
the  solution  is  not  sufficiently  active;  above  0.50  per  cent,  it  becomes  irritating. 

"Chemicals  Required  for  the  Preparation. — Three  chemical  substances  are 
indispensable  to  Dakin's  solution:  Chlorinated  lime,  anhydrous  sodium  carbon- 
ate and  sodium  bicarbonate.  Among  these  three  products,  the  latter  two  are 
of  a  practically  adequate  constancy,  but  this  is  not  the  case  with  the  first.  Its 
content  in  active  chlorine  (decoloring  chlorine)  varies  within  wide  limits,  and  it 
is  absolutely  indispensable  to  titrate  it  before  using  it. 

"Titration  of  the  Chlorinated  Lime. — There  must  be  on  hand  for  this  special 
purpose. : 

A  25-cc.  buret  graduated  in  o.i  cc. 

i  pipette  gaged  for  10  cc. 

A  decinormal  solution  of  sodium  thiosulphate  (hyposulphite).  This 
decinormal  solution  of  sodium  thiosulphate  can  be  obtained  in  the  market;  it 
can  also  be  prepared  by  dissolving  25  Gm.  of  pure  crystalline  sodium  thiosul- 
phate in  i  liter  of  distilled  water,  and  verifying  by  the  decoloration  of  an  equal 
volume  of  the  decinormal  solution  of  iodine  by  this  solution.  The  iodine  is 
prepared  by  dissolving  1.27  Gm.  iodine  and  5  Gm.  potassium  iodide  in  ioo\'cc.  of 
water. 

"The  material  for  the  dosage  thus  provided,  a  sample  of  the  provision  of 
chlorinated  lime  on  hand  is  taken  up  either  with  a  special  sound  or  in  small 
quantities  from  the  mass,  which  then  are  carefully  mixed. 

"  Weigh  out  20  Gm.  of  this  average  sample,  mix  it  as  completely  as  possible 
with  i  liter  of  ordinary  water,  and  leave  it  in  contact  for  a  few  hours,  agitating 
it  from  time  to  time.  Filter. 

"Measure  exactly  with  the  gaged  pipette  10  cc.  of  the  clear  fluid;  add  to 
it  20  cc.  of  a  i  :  10  solution  of  potassium  iodide  and  2  cc.  of  acetic  or  hydrochloric 
acid.  Drop,  a  drop  at  a  time,  into  this  mixture  a  decinormal  solution  of  sodium 
thiosulphate  until  decoloration  is  complete. 

"  The  number  of  cubic  centimeters  of  the  hypochlorite  solution  required  for 
complete  decoloration,  multiplied  by  1.775,  gives  the  weight  of  the  active  chlorine 
contained  in  100  Gm.  of  the  chlorinated  lime. 

"This  figure  being  known,  it  is  applied  to  the  accompanying  table,  which  will 
give  the  quantities  of  chlorinated  lime,  of  sodium  carbonate  and  of  sodium 
bicarbonate  which  are  to  be  employed  to  prepare  10  liters  of  Dakin's  solution. 

"Preparation  of  Dakin's  Solution. — To  prepare  10  liters  of  the  solution: 

1.  "Weigh  exactly  the  quantities  of  chlorinated  lime,  sodium  carbonate  and 
sodium  bicarbonate  which  have  been  determined  in  the  course  of  the  preceding 
trial. 

2.  "Place  in  a  1 2-liter  jar  the  chlorinated  lime  and  5  liters  of  ordinary  water, 
agitate  vigorously  for  a  few  minutes,  and  leave  in  contact  for  from  6  to  12 
hours,  over  night,  for  instance. 


STERILIZATION 


27 


QUANTITIES  OF  INGREDIENTS  FOR  TEN  LITERS  OF  DAKIN'S  SOLUTION 


Titer  of 
chlorinated  lime 

Chlorinated  lime, 
Gm. 

Anhydrous  sodium 
carbonate,  Gm. 

Sodium  bicarbonate, 
Gm. 

2O 

230 

H5 

96 

21 

22O 

110 

92 

22 

2IO 

105 

88 

23 

200 

IOO 

84 

24 

IQ2 

96 

80 

25 

I84 

92 

76 

26 

177 

89 

72 

27 

170 

85 

70 

28 

164 

82 

68 

29 

159 

80 

66' 

30 

154 

77 

64 

31 

148 

74 

62 

32 

144 

72 

60 

33 

I4O 

70 

59 

34 

135 

68 

57 

35 

132 

66 

55 

36 

128 

64 

53 

37 

124 

62 

52 

3.  "At  the  same  time  dissolve,  cold,  in  the  5  other  liters  of  water  the  sodium 
carbonate  and  the  bicarbonate. 

4.  "Pour  all  at  once  the  solution  of  the  sodium  salts  into  the  jar  containing 
the  maceration  of  chlorinated  lime,  agitate  vigorously  for  a  few  moments,  and 
leave  it  quiet  to  permit  the  calcium  carbonate  to  settle  as  it  forms.     At  the  end 
of  ]/2  hour  siphon  the  liquid,  and  filter  it  through  double  paper  to  obtain  an 
entirely  limpid  product,  which  must  be  protected  from  light. 

"Light,  in  fact,  alters  quite  rapidly  solutions  of  hypochlorite,  and  it  is  in- 
dispensable to  protect  from  its  action  the  solutions  which  are  to  be  preserved. 
The  best  way  to  realize  these  conditions  is  to  keep  the  finished  fluid  in  large  wicker 
covered  demijohns  of  black  glass. 

"Titration  of  Dakin's  Solution. — It  is  a  wise  precaution  to  verify,  from  time 
to  time,  the  titer  of  the  solution.  This  titration  utilizes  the  same  material  and 
the  same  chemical  substances  as  are  used  to  determine  the  active  chlorine  in 
the  chlorinated  lime. 

"Measure  out  10  cc.  of  the  solution,  add  2  cc.  of  1:10  solution  of  potassium 
iodide,  and  2  cc.  of  acetic  or  hydrochloric  acid.  Drop,  a  drop  at  a  time,  into  this, 
mixture  a  decinormal  solution  of  sodium  thiosulphate  until  decoloration  is 
complete. 

"The  number  of  cubic  centimeters  employed  multiplied  by  0.03725  will 
give  the  weight  of  the  sodium  hypochlorite  contained  in  100  cc.  of  the  solution. 

"The  Test  for  the  Alkalinity  of  Dakin's  Solution. — It  is  easy  to  differentiate 
the  solution  obtained  by  this  procedure  from  the  commercial  hypochlorites  and 
from  Labarraque's  solution. 


28  MEDICAL  BACTERIOLOGY 

"Pour  into  a  glass  about  20  cc.  of  the  fluid,  and  drop  on  the  surface  a  few 
centigrams  of  phenolphthalein  in  powdered  form.  Dakin's  solution,  correctly 
prepared,  gives  absolutely  no  change  in  tint,  while  in  the  same  conditions 
Javelle  water  and  Labarraque's  fluid  give  an  intense  red  coloration  which 
indicates  in  the  latter  two  solutions  the  presence  of  free  caustic  sodium." 

Numerous  preparations  have  been  put  forth  to  replace  the  D akin-Carrel 
solution.  None  of  them  has  as  yet  stood  the  test,  except  the  one  recommended 
by  Duret.  His  preparation  depends  upon  the  liberation  of  chlorine  from  mag- 
nesium hypochlorite,  instead  of  a  sodium  salt.  This  solution  is  more  stable, 
isotonic  with  blood  serum,  and  as  it  has  no  alkali  entering  into  its  preparation, 
the  latter  will  be  free  from  such  contamination.  The  following  is  its  formula  as 
published  in  the  Journal  of  the  American  Pharmaceutical  Association  (page 
241,  March,  1917). 

Chlorinated  lime 28 .  oo  Gm. 

Magnesium  sulphate 18 . 20  Gm. 

Water IOOO.GO  Gm. 

The  two  salts  are  triturated  in  a  mortar  and  the  water  added  gradually  in  small 
portions.  The  solution  is  then  filtered  through  cotton-wool. 

Solution  of  eusol,  and  chloramine,  the  coal-tar  derivative  containing 
available  chlorine,  have  been  recommended  recently  by  Dr.  Dakin  as  valuable 
disinfectants. 

A  complete  report  of  the  use  of  chloramine  in  the  treatment  of  infected 
wounds  may  be  found  in  the  Journal  of  the  A.  M.  A.  July  7,  1917,  page  27. 

The  organic  disinfectants  are  by  far  more  numerous  and  indeed  countless. 

Ethyl  alcohol  is  one  of  the  most  common  disinfectants  of  this  class.  By 
many  it  is  regarded  of  little  value,  but  experiments  recently  carried  out  verify 
the  results  of  Harrington  and  Walker  (Boston  Medical  and  Surgical  Journal, 
vol.  148,  p.  548).  These  men  found  that  "unless  the  bacterial  envelope  contains 
a  certain  amount  of  moisture,  it  is  impervious  to  strong  alcohol,  but  dried  bac- 
teria in  contact  with  alcohol,  containing  from  30  to  60  per  cent,  of  water,  will 
absorb  the  necessary  amount  of  water  therefrom  very  quickly  and  then  the 
alcohol  itself  can  reach  the  cell  protoplasm  and  destroy  it."  Adding  small 
portions  of  chloroform,  ether  and  more  commonly  acetone  (these  acting  as  fat 
solvent)  is  commonly  practised. 

Coal  tar  has  furnished  many  substances  that  possess  disinfectant  properties. 
Much  doubt  still  prevails  as  to  the  efficiency  of  numerous  members  of  this 
class  on  the  basis  of  value  assigned  them  due  to  laboratory  tests,  especially 
since  many  of  them  have  proven  of  little  value  in  practice.  Though  this  be  a 
fact,  still  there  remains  a  large  number  which  are  available  and  possess  a  high 
disinfectant  value. 

Phenol,  the  most  commonly  used  organic  disinfectant,  is  commonly  employed 
as  a  wash  in  strengths  from  i  to  5  per  cent. 

Cresol,  ortho-,  meta  and  para,  the  mixture  of  the  three  gives  rise  to  the 
commonly  used  tricresol.  These  are  more  powerfully  germicidal  than  phenol. 


STERILIZATION  29 

Such  antiseptic  value,  freedom  from  irritation  and  a  low  toxicity  tend  to  make  it 
valuable.  Lysol  and  liquor  cresolis  compositus  (U.S. P.)  are  preparations  of 
this  chemical  in  a  neutral  soap,  which  when  dissolved  in  water  form  a  clear  or 
slightly  opalescent  liquid.  Creolin  is  a  similar  preparation  with  a  resin  soap, 
forming,  however,  a  turbid  emulsion  with  water. 

There  are  numerous  other  coal-tar  disinfectants,  which  are  more  germicidal 
than  phenol.  They  are  valuable  deodorants  by  destroying  the  putrefactive 
bacteria  producing  such  odors.  They  are  less  toxic,  not  readily  affected  by 
organic  matter  and  much  cheaper  if  we  compare  their  relative  cost  and  disin- 
fectant coefficient  with  that  of  phenol. 

Formaldehyde  is  used  principally  as  a  gaseous  disinfectant.  A  solution  of 
the  gas  in  water  (liquor  formaldehydi,  U.S. P.)  when  used  as  a  liquid  disinfectant 
is  inferior  to  phenol  or  any  of  the  coal-tar  products. 

lodoform  in  itself  is  a  very  weak  antiseptic,  but  when  applied  to  wounds,  the 
organic  matter  present  effects  the  liberation  of  iodine,  which  results  in  bac- 
tericidal action. 

When  certain  dyes  were  discovered  to  possess  a  specific  staining  effect  for 
bacteria,  numerous  attempts  were  made  to  use  them  as  germicidal  or  antiseptic 
agents.  Drigalski  and  Conradi  observed  the  inhibitory  effect  that  crystal  violet 
possessed  upon  the  growth  of  the  cocci,  while  this  is  also  considered  when  in- 
troducing gentian  violet  in  the  preparation  of  Petroff's  media.  The  antiseptic 
value  displayed  by  brilliant  green,  crystal  violet,  and  malachite  green  can  be 
observed  in  the  chart  on  the  following  page,  as  published  by  Browning,  Henaway, 
Gulbrausen  and  Thornton.  Experiments  thus  far  performed  led  many  to  be- 
lieve that  the  dyes  possess  a  very  low  toxicity  and  at  the  same  time  a  high  anti- 
septic value. 

Flavine,  a  substance  belonging  to  the  latter  class,  has  recently  been  exten- 
sively exploited  by  the  above  four  co-workers,  at  the  Bland-Sutton  Institute  of 
the  Middlesex  Hospital  in  England,  their  original  work  having  been  published 
in  the  British  Medical  Journal  (No.  2925,  Jan.  20,  1917).  Flavine  is  a  substance 
belonging  to  the  acridine  group,  being  chemically  the  chloride  of  diamino- 
methyl  acridinum.  It  is  a  stable  chemical,  the  solutions  of  which  are  not  al- 
tered at  boiling  temperature. 

"  Flavine  has  been  found  to  possess  extremely  powerful  bactericidal  and 
antiseptic  properties,  which  are  enhanced  rather  than  diminished  by  admixture 
with  serum.  In  this  respect,  flavine  differs  from  all  other  antiseptics  in  common 
use. 

2.  In  the  presence  of  serum,  flavine  is  the  most  potent  bactericide  of  all  those 
investigated  for  both  staphylococcus  and  B.  coli  and  it  is  easily  efficient  for  the 
enterococcus  and  for  anaerobes,  such  as  B.  cedematis  maligni. 

3.  Flavine,  in  its  relation  to  its  bactericidal  power,  is  very  much  less  detri- 
mental to  the  process  of  phagocytosis   and  less  harmful  to  the  tissues  than 
the  other  substances;  hence  much  higher  effective  concentrations  can  be  em- 
ployed without  damaging  the  tissues  or  interfering  with  the  natural  defensive 
mechanisms. 


30  MEDICAL  BACTERIOLOGY 

4.  Clinical  results  have  substantiated  the  estimate  of  the  therapeutic  value 
of  flavine." 

The  following  chart  shows  the  antiseptic  value  of  flavine  and  other  substances, 
as  published  by  the  same  experimenters. 

GASEOUS  DISINFECTION 

The  best  and  most  ideal  way  of  disinfecting  and  destroying  those  agents 
that  produce  disease  is  by  the  use  of  a  suitable  gaseous  disinfectant.  Rooms, 
buildings  and  similar  compartments  could  not,  even  with  difficulty,  have  germi- 
cidal  solutions  applied  to  them,  for  it  would  be  hard  to  reach  all  surfaces  and 
then  hold  such  solutions  in  contact  for  a  sufficient  length  of  time,  in  order  to 
obtain  the  desired  bactericidal  action.  In  addition,  various  substances  would 
be  injured,  if  any  but  a  gaseous  disinfectant  be  used  as  a  means  of  obtaining 
sterility. 

The  most  practical  and  yet  a  suitable  and  efficient  gas  for  general  applica- 
tion is  formaldehyde.  It  is  virtually  non-poisonous,  non-corrosive,  non-in- 
jurious to  all  substances,  possesses  no  bleaching  effect  and  still  is  very  effective. 
There  are  numerous  methods  of  generating  this  gas,  to  be  used  in  practical 
operations;  but  this  should  be  noted,  when  using  any  of  the  methods. 

Formaldehyde  to  be  effective  must  be  used  in  an  environment,  the  tempera- 
ture of  which  should  not  be  below  6o°F.,  a  higher  temperature  is  more  preferable. 
With  the  gas  there  should  also  be  an  evolution  of  water  vapor,  producing  a 
relative  humidity  of  70  per  cent,  or  over.  Unless  the  conditions  be  adhered  to, 
it  is  useless  to  attempt  disinfection  with  formaldehyde,  as  otherwise  the  latter 
will  polymerize,  precipitating  a  solid  substance,  commonly  known  as  paraform 
or  paraformaldehyde.  In  all  cases  where  formaldehyde  disinfection  is  per- 
formed, all  cracks  should  be  hermetically  sealed  (especially  those  of  doors, 
windows,  etc.)  and  all  drawers  and  cupboards,  containing  fabrics  and  all 
apparel  should  be  freely  opened  and  the  latter  spread  about  to  permit  sufficient 
exposure  to  the  gas. 

One  of  the  first  methods  of  liberating  formaldehyde  gas  was  the  method  de- 
vised by  Trilatt,  in  which  he  directed  the  evaporation  of  the  gas  from  a  solution 
of  formalin  (a  40  per  cent,  solution  by  weight  of  the  gas  in  water)  to  which  he 
added  from  1 5  to  20  per  cent,  of  calcium  chloride.  Such  mixture  forms  a  prepara- 
tion, commonly  termed  formochloral,  which  possesses  the  characteristic  property 
of  practically  eliminating  polymerization.  Glycerin  has  been  suggested  by 
Schlossmann,  in  concentration  of  10  per  cent,  as  a  substance  replacing  calcium 
chloride  and  hindering  polymerization.  The  apparatus  used  for  the  latter  and 
similar  solutions  consists  of  a  generator,  which  permits  the  solution  contained 
therein,  when  heated,  to  flow  in  a  fine  stream  through  a  copper  coil  heated  to 
redness  by  a  flame,  the  gas  vapor  then  passing  directly  into  the  room  together 
with  some  moisture  in  a  superheated  and  effective  condition.  Such  generators 
can  be  operated  outside  the  room  to  be  disinfected. 

Similar  to  the  latter  and  a  most  efficient  method  of  disinfecting  with  for- 
maldehyde is  the  generation  of  the  gas  within  an  autoclave  set  to  blow  off  at 


STERILIZATION  3 1 

45  pounds  pressure.  The  apparatus  is  set  up  outside  the  room  or  compartment 
to  be  treated  and  a  hose  attached  to  the  autoclave  delivers  formaldehyde-laden 
steam  through  an  opening. 

Blankets  or  sheets  immersed  and  saturated  with  a  solution  of  formalin  have 
been  recommended  to  be  hung  in  and  about  the  room  to  be  disinfected,  to  allow 
the  liberation  of  the  gas  by  the  heat  present.  To  displace  this,  some  have  pro- 
posed spraying  the  room  with  formalin  from  a  compressed-air  or  steam  atomizer. 
Neither  of  these  methods  is  certain  as  to  the  liberation  of  gas  and  the  operator 
must  be  careful  and  work  rapidly,  as  formalin  is  irritating  to  the  conjunctiva 
and  other  mucous  membranes. 

A  most  efficient  and  convenient  method  used  is  the  liberation  of  the  gas 
within  the  room  to  be  disinfected.  The  method  used  with  universal  approval 
is  the  permanganate  method.  Potassium  permanganate  when  mixed  with 
formalin  results  in  an  active  reaction  with  the  evolution  of  heat,  which  in  turn 
causes  the  evaporation  of  formaldehyde  gas  together  with  water  vapor.  While 
some  of  the  gas  is  actually  consumed  in  the  violent  reaction,  the  yield  is  more 
than  by  other  methods  and  preferable  because  of  the  quick  and  sudden  evolution 
of  all  the  gas,  together  with  some  moisture. 

In  practice,  i  quart  of  formalin  is  poured  over  i  pound  of  permanganate 
crystals,  using  such  proportions  for  each  1000  cubic  feet  of  space.  The  operator 
must  be  cautioned  not  to  throw  the  permanganate  into  the  formalin,  for  an 
explosion  will  result.  On  account  of  the  vigorous  ebullition  and  foaming  that 
takes  place,  high  cylindrical  vessels  should  be  used,  about  10  or  12  inches  in 
height,  which  possess  flared  or  funnel-like  tops,  so  as  to  further  assure  any  of 
the  sputtered  material  from  being  thrown  out.  A  small  lo-quart  milk  pail 
answers  well  for  such  a  purpose. 

Numerous  modifications  have  been  attempted  recently  to  use  other  chemicals 
instead  of  the  permanganate  salt.  Potassium  and  more  recently  sodium  dichro- 
mate  have  replaced  the  latter,  due  to  the  cheapness  in  cost  of  material.  Others 
have  used  lime  or  quicklime,  in  which  method  the  gas  was  generated  by  pouring 
i  pint  of  the  solution  of  formalin  over  quicklime  (from  J^  to  i%  pounds)  con- 
tained in  a  wide  shallow  pan,  placed  in  a  basin  of  water. 

The  dangers  and  inconveniences  experienced  with  the  foregoing  methods 
may  be  avoided  if  we  use  the  " so-called"  formaldehyde  candles  or  other  forms 
of  solid  formaldehyde,  as  the  source  from  which  to  obtain  the  gas.  The  Sherring 
lamp  was  the  first  apparatus,  in  which  solid  formaldehyde  or  paraform  was  used 
as  a  means  from  which  to  generate  the  gas.  Heat  was  obtained  from  the  igni- 
tion of  alcohol,  which  in  turn  decomposed  the  paraformaldehyde,  liberating 
gaseous  formaldehyde.  A  temperature  of  at  least  275°?.  is  required  for  the 
active  generation  of  this  gas  from  paraform. 

All  other  forms  of  solid  formaldehyde  and  paraform  candles  are  used  the 
same  way,  by  applying  heat  to  these  substances,  contained  in  metal  receptacles. 
Two  ounces  of  paraformaldehyde  generates  sufficient  gas  to  insure  efficient 
disinfection  of  a  room  having  a  capacity  of  1000  cubic  feet. 

Numerous  suitable  cabinets  have  been  recently  made  and  are  in  use  by 


32  MEDICAL  BACTERIOLOGY 

pharmacists,  sanitary  barber  shops  and  other  dealers  for  the  disinfection  of 
shaving,  hair,  tooth  and  other  brushes.  Paraform  is  here  used  as  the  generator 
and  the  gas  passing  through  wire  shelves  can  readily  come  into  contact  with  all 
articles  placed  therein. 

The  value  of  gaseous  formaldehyde  as  a  germicide  has  from  time  to  time  been 
disputed.  This  question  has,  however,  been  recently  settled  by  a  discussion, 
in  which  numerous  authorities  and  health  officers  entered;  all  favoring  a  con- 
tinuance of  formaldehyde  gas  disinfection  (see  Williams,  "  Aerial  or  Gaseous 
Disinfection,"  Journ.  of  the  American  Pharmaceutical  Association,  March, 


Sulphur  has  been  extensively  used  for  the  generation  of  SO2  gas,  which  is 
an  efficient  germicide.  In  order  to  be  effective,  water  must  be  vaporized,  since 
the  disinfecting  action  takes  place  upon  the  formation  of  sulphurous  acid  by  the 
gas  with  water.  From  3  to  3^  pounds  of  sulphur  should  be  burned  for  each 
1000  cubic  feet  of  space  and  the  gas  be  allowed  to  remain  in  contact  for  at  least 
24  hours.  This  gas  is  an  active  bleaching  agent.  It  is  perhaps  more  used  as 
an  insecticide  rather  than  a  germicide,  a  property  which  is  not  possessed  by 
formaldehyde.  When  used  for  killing  insects  and  vermin  water  need  not 
be  used  in  conjunction  with  it,  because  it  is  just  as  efficient  for  this  purpose  when 
dry,  and  thus  will  not  seriously  tarnish  or  bleach  the  contents  of  the  room. 

Don't  attempt  to  use  formaldehyde  and  sulphur  at  the  same  time,  the  latter 
as  an  insecticide  and  the  former  as  a  germicide.  Instead  of  acting  as  synergists 
they  seem  to  oppose  each  other. 

Oxygen  in  its  nascent  state  is  an  active  bactericidal  agent.  It  is  this  gas 
that  performs  the  germicidal  action  when  chlorine  is  used  as  a  gaseous  disin- 
fectant. The  latter  acts  only  in  the  presence  of  moisture,  in  which  the  hy- 
drogen of  the  water  molecule  unites  to  form  HC1  with  the  chlorine,  liberating 
the  nascent  oxygen.  The  latter  then  does  the  disinfection.  This  gas  practic- 
ally is  inadequate  because  of  its  active  bleaching  and  injurious  properties  upon 
all  materials. 

Bromine  has  been  used  both  as  an  insecticide  as  well  as  a  germicide,  but  is 
so  poisonous  as  to  be  too  dangerous  for  ordinary  use. 

Hydrocyanic  gas  is  used  as  an  insecticide.  It  is  powerful  enough  to  kill 
insects  but  has  no  effect  upon  foliage.  When  used,  the  trees,  foliage  and  other 
environment  are  covered  to  keep  in  the  vapor  and  the  gas  is  generated  from 
KCN  and  dilute  HC1,  being  kept  in  over  night.  This  gas  is  rarely  used  for  any 
purpose,  other  than  the  disinfection  of  foliage,  because  of  its  deadly  poisonous 
properties. 

For  years  the  apothecary  has  been  directed  to  have  present  in  the  con- 
tainers of  the  various  drugs,  small  vials  containing  chloroform,  ether  or  other 
such  volatile  substances,  which  because  of  their  antiseptic  character,  are  both 
detrimental  to  the  propagation  of  bacteria  and  perhaps  more  so  to  the  life  of  the 
various  parasites  these  drugs  harbor.  Notable  examples  of  such  drugs  are  ergota 
linums,  etc.  The  various  "waters"  of  the  United  States  Pharmacopeia  may 
be  kept  for  a  greater  length  of  time  by  adding  to  such  containers  a  few  drops  of 


STERILIZATION 


33 


Bacillus  coli  communis 

pi! 

£8,8-3 

rt 
0 

N 

O 

<N 
0 

0 

;r 

H 

0 

t- 

1 

10 

0 

0 

H 

1 

lethal  concentration  in  serum 
Antisentie  not^nrv  ^  lethal  concentration  of  substance  in  question 

lethal  concentration  of  chloramine-T  in  serum  (taken  as  one) 
*KI  in  the  concentration  employed  in  the  solution  of  Iodine  by  itself  produces  no  bactericidal  action. 

In  serum 

Si 

W             Tj- 

in      -*t 

0 
0 

0 

" 

0 

t 

* 

0 

o 

* 

Lethal 
concen- 
tration 

0        § 
in      o 

m     o 

0 

vo       O 

N  § 

IOOO 

sufficient 

§ 

10,000 

O 

o 

§ 

1 

§ 

1 

i 

I 

"Z 

o  5 

l! 

05  C 
fe   «> 
f   0 

G 

h-l 

'-£  '£>  G 

c  a_« 

00          <N 

ro 

m     \o 

0             M 

0 

0 
0 

o 

" 

§ 

o 

-t 

o 

0 

10 

| 

o 

CO 

rg 

; 

m 

Lethal 
concen- 
tration 

§        § 
O        O 

(M          00 

0 

o 

ro        O 

c,     1 

§ 

0 

o 
m 

ooo'ooo'i  : 

o 

o 

0 

:  130,000 

I 

:  20,000 

00 

0 

§ 

I  ~ 

Staphlococcus  aureus 

|l|| 

IO                            1/5            • 

0                         0           •              0           • 

\ 

« 

1/3 

| 

0 

H 

0 

Concentra- 
tion which 
inhibits 
phagocy- 
tosis 

m      § 

(N          IO 

§ 

0,      1 

§ 

IO 

M 

0 

o 

I 

o 

o 

§ 

I 

I 

! 

o 
o 

Lethal  concentration  =  that  which  suffices  to  kill  organisms  as  tested  by  subcultures. 
Theraoeutie  ropffiripnt  -  ri+ir,  concentration  which  reduces  phagocytosis  by  50  per  cent. 

2 
c 

1 

Lethal  Anti- 
concen-  septic 
tration  potency 

in       rj- 

0 

6 

o 
o 

0) 

o 

<N 

M 

1 

1 

1 

90 

1 

o 

0        0 

,s 

m     o 

<N          O 
0 

1000 

sufficient 

Q 

10,000 

0 

0 

30,000 

100,000 

j 

} 

(N 

i 

M            M 

M          M 

1 

SI 

ll 

f-   0 

C 
1—  1 

Anti- 
septic 
potency 

00          <N 
ro 

m     o 

0            M 

0 

ro       M 
0 

0 

0 

" 

1 

* 

I 

40,000 

a 

o 

0 
0 

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•o 

0 

0 

OO 

Lethal 
concen- 
tration 

0        0 
O        O 
0        0 
<N         00 

ro        O 

§ 

1 

To 

| 

:  10,000 

1 

o" 

0* 

o 

o 

§ 

rf 

1 

:  20,000 

Antiseptic 

Chloramine-T  (content  f  Concentration  of 
in  "available"  Cl  =  f  substance  
25  per  cent.  )  Concentration  of 
("available"  Cl.  .  . 

E  u  s  o  1  (content  in  f  Dilutions  of  stan- 
"  available"  Cl  =  0.34]  dard  solution  
per  cent.  )  Concentration  of 
("available"  Cl.  .  . 

Dakin's  sol.  (modified  Dilutions  of  stan- 
by  Daufresne  =  0.22  dard  solution  
per  cent,  "available"  Concentration  of 
Cl).  "available"  Cl.  .  . 

Chlorine  water  (concentration  of  "Cl")  

Carbolic  acid  

Mercury  perchloride  

0 

1 

n  sulphate  

>n  oxalate  

een  oxalate  and  sulphate. 

iiprein  hydrochloride  

Flavine  

Brilliant  gree 

Brilliant  grec 

Malachite  gr 

O 

Ethylhydroc 

34  MEDICAL  BACTERIOLOGY 

chloroform  or  ether.  The  small  quantity  of  antiseptic  so  added  is  not  detri- 
mental or  observable  either  during  dispensing  or  compounding,  but  yet  sufficient 
to  prevent  the  growth  of  moulds  and  other  bacteria. 

Inasmuch  as  the  retail  pharmacist  today  is  called  upon  to  dispense  numerous 
preparations  and  medicaments  as  sterile  products,  it  would  be  advisable  to 
note  what  medicines  require  sterilization.  These  may  be  grouped  as  follows: 

(a)  Those    preparations    intended    for    intravenous,    intramuscular    and 
hypodermic  use,  which  must  be  absolutely  sterile. 

(b)  Drops,  lotions,  ointments,  and  other  applications  to  be  used  for  the 
eye. 

(c)  All  preparations  to  be  applied  to  the  bruised  or  abrased  skin  and  gener- 
ally in  other  cases  when  the  latter  is  broken  and  medicaments  are  to  be  applied 
thereon. 

(d)  Solutions  to  be  used  for  inflamed  mucous  surfaces  and  for  irrigation  of 
various  internal  organs. 

(e)  All  preparations  that  become  mouldy,  which  in  turn  may  affect  their 
composition,  as  liquor  magnesii  citratis  (U.S. P.). 

Many  of  the  products  that  the  pharmacist  is  called  upon  to  dispense  are 
extemporaneous  preparations,  to  be  dispensed  as  quickly  as  possible.  It  is 
perhaps  true  that  some  of  these  cannot  be  made  absolutely  sterile  in  the  short 
time  available,  but  all  of  .them  can  be  made  as  near  sterile  as  is  essential  for 
ordinary  administration. 

Ointments. — All  bases  and  vehicles  can  be  kept  sterile  indefinitely  if 
the  original  product  when  first  made  be  kept  in  a  clean,  sterile  container  in  a 
cool  place;  and  each  time  a  portion  is  to  be  removed,  a  spatula,  which  has  been 
cleansed  with  a  little  alcohol  or  chloroform,  should  be  used. 

Most  of  the  powders  entering  into  the  composition  of  an  ointment,  if  kept 
in  a  clean-stoppered  container  will,  with  few  exceptions,  not  require  any  addi- 
tional means  to  produce  a  sterile  product. 

In  compounding,  use  a  clean  slab  and  spatula,  which  have  been  previously 
cleansed  with  alcohol  or  chloroform.  Place  the  end  product  preferably  in  a 
collapsible  tube,  a  supply  of  which  can  easily  be  kept  on  hand,  at  all  times,  in  a 
jar  containing  alcohol,  to  assure  sterility. 

Liquid  Preparations. — Use  sterile  containers  and  stoppers.  Use  sterile 
water  and  have  all  other  solutions  sterile  if  convenient,  when  these  enter  into 
the  preparation  of  any  medicinal  product  which  is  to  be  sterile. 

If  such  preparations  are  not  rendered  inactive  or  have  their  composition 
otherwise  affected,  place  them  either  in  the  autoclave,  steam  sterilizer  or  water 
bath  for  a  sufficient  length  of  time. 

In  those  instances  where  fractional  sterilization  is  recommended,  heating  for 
one  period  will  suffice  for  all  such  extemporaneous  preparations. 

Sterilization  by  heat  may  be  accomplished  by  exposure  to  fire,  by  hot  air, 
hot  water  or  steam. 

Small  instruments  which  can  be  easily  and  entirely  flamed,  without  injury, 
may  be  conveniently  sterilized  in  this  way.  Larger  instruments,  utensils, 


STERILIZATION  35 

empty  flasks,  jars,  bottles,  tubes  and  pipettes,  are  commonly  sterilized  in  a  hot- 
air  sterilizer.  They  can,  if  necessary,  be  sterilized  by  boiling  or  steaming,  but 
ordinarily,  hot  air  is  preferable. 

Culture  media  and  pharmaceutical  preparations  as  a  whole  cannot  be 
sterilized  by  hot  air  without  destruction. 

Hot-air  sterilizers  are  constructed  to  maintain  an  even  distribution  of  heat 
throughout,  and  are  equipped  with  thermometers.  It  is  a  safe  rule,  especially 
when  sterilizing  thick  glass  and  inflammable  material,  to  put  the  articles  in  the 
sterilizer  before  it  has  been  heated,  to  elevate  the  temperature  gradually.  After 
sterilization  allow  the  sterilizer  and  its  contents  to  cool  to  room,  temperature 
before  opening  the  sterilizer. 


FIG.  4. — LAUTENSCHLAGER  HOT-AIR  STERILIZER.    - 

Hot  air  lacks  the  penetrating  power  of  steam  and  at  the  same  temperature, 
hot  air  is  less  germicidal  than  steam.  Exposure  to  a  temperature  of  i5o°C. 
for  i  hour  or  2oo°C.  for  30  minutes  in  the  hot-air  sterilizer  is  necessary  to  affect 
the  destruction  of  bacteria.  Spores  frequently  withstand  this  and  to  destroy 
them  a  temperature  of  i5o°C.  must  be  maintained  for  several  hours  or  2oo°C. 
for  2  hours. 

The  quickest  and  most  sure  method  of  sterilization  is  accomplished  by  means 
of  an  autoclave.  An  autoclave  is  a  vertical  or  horizontal  cylinder,  constructed 
like  a  boiler.  There  is  a  gas  burner  beneath  it  for  heating,  and  a  water  jacket 
several  inches  deep  at  the  bottom.  A  perforated,  false  bottom,  rises  above  the 
water.  An  air-tight  door  or  lid  on  one  end  opens  to  permit  loading  and  un- 
loading. This  lid  is  fastened  in  place  with  a  number  of  thumb  screws.  It  is 
equipped  with  a  dial  which  registers  pounds  pressure,  and  two  valves,  an  exhaust 
valve  and  a  safety  valve.  The  safety  valve  is  set  to  blow  off  steam  at  15  pounds 
pressure.  To  operate  one  must  first  see  that  sufficient  water  is  in  the  autoclave 
(several  inches);  the  substance  to  be  sterilized  is  then  placed  in  the  autoclave 
and  the  lid  fastened  tight  with  the  thumb  screws.  The  exhaust  valve  is  opened 


MEDICAL  BACTERIOLOGY 


and  the  burner  under  the  autoclave  lighted.  As  the  autoclave  becomes  hot  the 
contained  air  expands  and  escapes  through  the  open  valve,  then  steam  forms, 
eventually  all  the  air  is  expelled  and  steam  begins  to  escape.  When  steam  comes 
out  of  the  exhaust  valve,  it  is  closed,  then  the  pressure  within  rises  until  it 
registers  15  pounds  on  the  dial.  As  soon  as  the  pressure  reaches  15  pounds  the 
time  is  noted  and  exactly  20  minutes  later  the  flame  beneath  the  autoclave  is 
turned  out.  Gradually  the  pressure  falls  as  the  autoclave  cools.  It  is  not 

opened  until  the  needle  on  the  dial  falls  to 
the  zero  mark.  Then  the  contents  of  the 
autoclave  are  removed,  sterile.  An  exposure 
to  steam  under  15  pounds  pressure  for  20 
minutes,  kills  spores  as  well  as  bacteria;  it 
achieves  complete  sterility. 

This  is  the  method  of  choice  for  steriliz- 
ing water,  salt  solution,  bouillon,  agar,  milk, 
camphorated  oil,  mercury  salicylate,  mercury 
sozoiodolate  and  other  substances  which  are 
not  injured  by  so  high  a  temperature,  i2o°C. 
When  an  autoclave  is  not  available  and  a 
substance  suitable  for  autoclave  sterilization 
must  be  sterilized  at  once,  make  brine  by 
adding  common  salt  to  watef,  place  in  a 
water  bath  or  boiler,  immerse  the  substance 
to  be  sterilized  and  boil  from  30  to  60  min- 
utes. The  addition  of  salt  to  water  elevates 
its  boiling  point  close  to  the  temperature  of 
the  autoclave  at  15  pounds  pressure. 

Steam  sterilization  without  pressure  is 
usually  done  in  an  "Arnold"  or  similar 
sterilizer,  an  apparatus  having  a  square 
metal  pan  about  4  inches  deep,  in  the 
center  of  which  is  an  upright  cylinder  about 
inches  high,  that  opens  into  a  large 


5. — AUTOCLAVE. 


and 
This 


6    inches    in     diameter 

cubical  compartment.  This  upper  compartment,  in  which  sterilization 
is  affected,  has  double  walls  and  an  inner  and  an  outer  door.  The 
apparatus  is  so  constructed  that  when  the  pan  is  filled  with  water,  a 
flame  placed  beneath  it  and  steam  generated,  the  steam  escapes  through 
the  cylinder  in  the  center  of  the  pan  into  the  sterilizing  compartment 
above.  Anything  that  can  be  sterilized  in  an  autoclave  without  injury,  may  be 
sterilized  in  the  "Arnold"  steam  sterilizer  by  steaming  it  i  hour  each  day  for 
3  consecutive  days.  This  method  of  sterilizing  by  short  exposure  each  day,  for 
a  number  of  days,  is  called  intermittent,  discontinuous  or  fractional  steriliza- 
tion. It  is  based  on  the  theory  that  steam  at  ioo°C.  will  kill  all  bacteria  in 
i  hour,  but  not  spores,  the  spores  surviving  the  first  hour  of  sterilization,  de- 
velop, in  the  following  hours,  into  bacteria,  and  as  bacteria,  are  killed  when 


STERILIZATION 


37 


steamed  the  second  day.  The  third  exposure  killing  bacteria,  developing  from 
spores  that  might  have  escaped  the  first  two  exposures.  For  spores  to  develop 
into  bacteria,  conditions  favorable  to  growth  must  prevail  during  the  periods 
between  steamings.  The  material  upon,  or  in  which  the  spores  are  lodged,  must 
contain  available  bacterial  food  and  the  temperature  must  be  about  2o°C.  to 
35°C.  It  will  be  seen  that  intermittent  or  fractional  sterilization  is  not  adapt- 
able to  sterilization  of  instruments,  etc. 

Quite  a  number  of  culture  media  and  many  pharmaceuticals  undergo 
chemical  changes  and  are  injured 
by  autoclaving,  boiling  in  brine  or 
steaming  for  an  hour.  These  are 
sterilized  according  to  the  degree  of 
heat  they  can  withstand.  Some  are 
placed  in  the  steam  sterilizer  for  J^ 
hour  for  3  consecutive  days,  others 
are  given  20  minutes  or  10  minutes 
each  day  for  3  consecutive  days. 
Preparations  that  cannot  be  exposed 
to  a  temperature  of  ioo°C.  for  any 
time  without  injury,  are  sterilized 
by  heating  them  in  a  water  bath  at 
90°,  80°,  70°  or  6o°C.  from  15 
minutes  to  i  hour  each  day  for  5 
or  6  consecutive  days,  according  to 
their  degree  of  stability. 

Since  some  spores  and  a  few 
bacteria  can  resist  temperatures 
below  the  boiling  point  for  many 
hours,  to  insure  the  sterility  of  preparations  that  cannot  be  heated 
above  80°  or  9o°C.,  ingredients  that  may  be  autoclaved  or  steamed,  are 
first  sterilized  that  way.  Sterile  utensils  are  used  throughout  preparation  and 
care  taken  to  avoid  contamination  by  air,  etc.  As  an  example,  let  us  consider 
the  preparation  of  litmus  lactose  gelatin.  A  saturated  aqueous  solution  of 
litmus  is  made,  also  plain  bouillon;  these  are  separately  sterilized  in  autoclave. 
Gelatin  which  has  been  kept  sealed  in  dust-proof  packages  to  prevent  possible 
contamination  is  dissolved  in  sterile  bouillon,  sterile  litmus  added,  and  then  i 
per  cent,  of  lactose,  the  lactose  having  been  kept  in  an  air-tight  container  and 
weighed  out  on  a  sterile  watch  glass.  By  means  of  a  sterile  test-tube  filler,  the 
litmus  lactose  gelatin  is  run  into  cotton-stoppered  test-tubes  or  flakes  previously 
sterilized  in  a  hot-air  sterilizer.  This  litmus  lactose  gelatin,  which  cannot 
withstand  steaming  for  prolonged  periods,  which  has  been  guarded  against  con- 
tamination and  made  of  sterile  ingredients,  so  far  as  possible,  usually  contains 
few  bacteria  and  fewer  spores.  It  is  placed  in  the  steam  sterilizer  for  15  minutes 
each  day  on  3  consecutive  days,  being  kept  at  room  temperature  between 
steamings.  In  nearly  all  cases  complete  sterility  is  produced. 


FIG.  6. — ARNOLD  STEAM  STERILIZER. 


38  MEDICAL  BACTERIOLOGY 


STERILIZATION  TABLES 

HOT  AIR 

Material  Temperature      Time,  hours 

Flasks,  bottles,  test-tubes,  etc.,  stoppered  with  cotton  or  wrapped 
in  paper 1 5o°C.  i  to  3 

Flasks,  petri  dishes,  tubes,  ampules,  pipettes  and  other  utensils, 
not  injured  by  heat,  not  stoppered  nor  wrapped  with  inflam- 
mable material '. 200°  to  3oo°C.  i  to  2 

Talc,  iso°C • i  to  3  hours 

Kaolin,  i5o°C i  to  3  hours 

Boric  acid,  i5o°C. i  to  3  hours 

Zinc  oxide,  i5o°C i  to  3  hours 

» 

(Powders  should  be  spread  in  a  stratum  not  more  than  %  inch  thick.) 

AUTOCLAVE — 15  POUNDS  PRESSURE  FOR  20  MINUTES 

Water Plain  bouillon 

Salt  solution Plain  agar 

Oils Glycerin  bouillon 

Milk Glycerin  agar 

Potato Mercury  sozoiodolate 

Mercury  Salicylate 

STEAM  (iooJC.) 

Material  Time,  minutes  Np.  of  days 

Gelatin 15  3 

Bouillon  and  agar  containing  sugar 15  3 

Sugars 15  3 

Litmus  milk 15  3 

Adrenalin 15  3 

Argyrol 15  3 

Arsenic 15  3 

Atropin 15  3 

Caffeine 15  3 

Calomel  cream 15  3 

Gray  oil 15  3 

Gums 15  3 

Mercury  benzoate 15  3 

Mercury  cacodylate 15  3 

Morphine 15  3 

Paraffin 15  3 

Sodium  cacodylate 15  3 

Stovaine 15  3 

Straphantin 15  3 

Endo's  medium 15  3 

MacConkey's  bile-salt  medium 10  a 

Alkaloids,  generally 10  2 


STERILIZATION 


39 


HOT-WATER  BATH  (60°  TO  8o°C.) 


Material 


Time,  hours 


Blood  serum 

Cacodylates 

Caffeine  benzoate. . 

Cocaine 

Duboisine 

Ergot 

Eserine  sulphate. . . 
Glycerophosphates . 

Hyoscine 

Quinine 

Physostigmine .... 

Scopalamine 

Strychnine 

Trypsin 


No.  of  days 

4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
4  to  6 
.  4  to  6 
4  to  6 
4  to  6 
4  to  6 


Heating  at  relatively  low  temperatures  (60°  to  8o°C.)  for  an  hour  each  day 
over  a  number  of  days  is  called  pasteurization.  It  insures  the  destruction  of 
over  90  per  cent,  of  bacteria  and  attenuation  of  those  not  destroyed;  so  much  is 
accomplished  by  the  first  heating.  Spores  resist  this  treatment  for  from  4 
to  5  days  sometimes.  While  pasteurization  continued  for  a  week  usually  steri- 
lizes, sometimes  it  fails  to;  therefore,  pasteurized  materials  should  be  tested  and 
examined  to  prove  sterility,  before  they  are  used.  This  is  imperative  when 
dealing  with  quinine  preparations  intended  for  hypodermic  or  intravenous 
administrations,  as  quinine  may  be  contaminated  with  tetanus  spores. 

Different  species  of  bacteria,  even  different  strains  of  a  single  species,  vary 
in  their  resistance  to  heat.  As  heating  tends  to  lessen  the  therapeutic  value  of 
bacterins  or  bacterial  vaccines,  the  temperature  and  time  of  exposure  must  be 
varied  so  as  to  produce  sterilization  with  the  minimum  temperature  and  time 
of  exposure  that  will  accomplish  it. 


STERILIZATION  OF  BACTERINS 

Organism  Temperature 

Typhoid 6o°C. 

Staphylococci 6o°C.  to  8o°C. 

Streptococci S5°C. 


Time,  hours 


5S°C. 

6o°C. 

Pneumococcus .  .  6o°C. 


M  eningococcus . 
Gonococcus. . . 


Micrococcus  catarrhalis 

Bacillus  of  influenza : 

Bacillus  of  diphtheria 

Bacillus  of  friedlander 

Bacillus  of  dysentery 

Bacillus  of  paratyphoid 

Bacillus  coli S5°C. 

Bacillus  pestis 5S°C. 

Spirillum  of  cholera 55°C. 

Sensitized  vaccines SS°C. 


55-°C. 
S5°C. 
6o°C.  to  7o°C. 

ss°c. 

S5°C. 
55°C. 


CHAPTER  VI 
CULTURE  MEDIA 

Culture  media  are  sterile  fluids  and  solids  upon  which  bacteria  are  cultivated 
to  determine  the  character  of  their  growth,  to  study  and  classify  species,  to  iso- 
late some  organisms  from  others,  to  detect  the  presence,  number  and  kind  of 
bacteria  in  water,  milk,  etc.,  and  to  obtain  large  numbers  of  certain  organisms 
for  the  preparation  of  diagnostic  and  therapeutic  agents,  such  as  bacterial 
vaccines. 

For  convenience  of  description,  culture  media  may  be  divided  into  two 
classes,  natural  and  artificial. 


NATURAL  CULTURE  MEDIA 
Fluid  Solid 


Milk 

Whey 

Blood  serum 

Serous  fluids 

Eggs 

Ox  bile 

Urine 


Blood  serum 

Eggs 

Potato 


ARTIFICIAL  CULTURE  MEDIA 


Fluid 


Bouillon 
Lactose  bouillon 
Glucose  bouillon 
Saccharose  bouillon 

With  or  without  litmus 
Other  sugar  bouillons 
Glycerin  bouillon 
Ox-bile  bouillon 
Dunham's  solution 
MacConkey's  bile-salt-medium 
Trypsinized-peptone-water 


Solid 


Agar 

Lactose  agar 
Glucose  agar 
Saccharose  agar 

With  or  without  litmus 
Other  sugar  agar  media 
Glycerin  agar 
Blood-smeared  agar 
Endo's  agar 
Russell's  agar 
Malachite-green  agar 
Gelatin  media 
PetrofFs  medium 


40 


CULTURE    MEDIA  41 

Some  bacteria  grow  luxuriantly  on  a  variety  of  media,  others  will  only  grow 
on  a  few  substances  especially  adjusted  to  their  requirements.  Hence,  we  have 
a  number  of  culture  media,  some  used  for  the  cultivation  of  bacteria  in  general 
and  others  for  the  cultivation  of  one  or  several  species. 

PREPARATION  OF  CULTURE  MEDIA 

Litmus  Milk. — Fresh,  sweet,  cow's  milk,  which  contains  not  more  than  3 
per  cent,  fat: 

1.  Add  aqueous  sol.  of  litmus,  sufficient  to  give  a  faint  blue  tint. 

2.  Sterilize  in  Arnold  steam  sterilizer,  i  hour  each  day  for  3  consecutive  days. 
Plain  Bouillon  (Meat  extract  broth) . 

1.  Beef  extract  (Liebig's),  2  Gm. 

2.  Beef  peptone  (Witte's),  10  Gm. 

3.  Sodium  chloride,  5  Gm. 

4.  Water,  1000  cc. 

Make  a  paste  of  the  peptone;  then  dissolve  it  in  water,  add  the  salt  and  beef 
extract.  Boil  and  stir  for  %  hour,  make  up  for  evaporation  by  the  addition  of 
water,  again  bring  to  boiling  point.  Test  for  alkalinity  or  acidity  and  properly 
adjust  the  reaction.  Filter  while  hot  and  again  when  cool.  Sterilize  in  auto- 
clave. 

Plain  Bouillon  (Meat  infusion  broth). 

1.  Minced  lean  meat  (beef  or  veal),  500  Gm. 

2.  Water,  1000  cc. 

Place  in  ice  box  in  shallow  dish  for  24  hours.  Skim  off  fat,  filter  through 
linen  and  express  juice  contained  in  the  meat. 

Boil  for  2  hours,  filter  through  linen  and  boil  again  for  i  hour.  Add  water 
to  make  up  for  loss  by  evaporation,  add  peptone  10  Gm.  and  sodium  chloride 
5  Gm.,  boil  for  J^  hour,  make  up  for  evaporation,  adjust  reaction,  filter  while 
hot  and  again  when  cool.  Sterilize  in  autoclave. 

Sugar-free  Bouillon. — Make  meat  infusion  broth,  introduce  bacillus  coli 
and  incubate  for  24  hours  at  37°C.,  boil  for  J^  hour,  filter  while  hot  and  again 
when  cool.  Adjust  reaction  and  sterilize. 

Sugar  Bouillon. — Various  sugars  are  added  to  plain  bouillon  or  stigar-free 
bouillon  to  study  fermentation.  Glucose,  lactose  and  saccharose  are  most 
commonly  used.  J^  to  2  Gm.  to  100  cc.,  J^  to  2  per  cent. 

Bouillon  containing  sugar  is  often  tinted  a  faint  blue  by  adding  litmus;  this 
is  to  act  as  an  indicator  of  acid  production. 

Culture  media  containing  sugars  are  sterilized  in  the  Arnold  steam  sterilizer 
(ioo°C.)  20  minutes  each  day  for  3  days. 

Glycerin  Bouillon. 

1.  Plain  bouillon,  100  cc. 

2.  Double  distilled  glycerin,  6  cc. 
Sterilize  in  autoclave. 


42  MEDICAL  BACTERIOLOGY 

Calcium  Carbonate  Bouillon. — Sterilize  powdered  calcium  carbonate  or 
marble  chips  in  hot-air  sterilizer  at  i5o°C.  for  2  hours. 

1.  Plain  bouillon  (meat  infusion),  100  cc. 

2.  Calcium  carbonate,  i  Gm. 

3.  Glucose,  i  Gm. 

Sterilize  in  steam  sterilizer  at  ioo°C.  20  minutes  each  day  for  3  days. 

Dunham's  Solution  (Peptone-salt-sol.). 

1.  Peptone  (Witte's),  10  Gm. 

2.  Sodium  chloride,  5  Gm. 

3.  Distilled  water,  1000  cc. 

Boil  15  to  30  minutes,  add  water  to  make  up  for  evaporation,  filter  while 
hot  and  again  when  cold. 

Sterilize  in  steam  sterilizer  at  ioo°C.  20  minutes  each  day  for  3  days. 

Trypsinized  Peptone-water,  (Rivas). 

1.  Dissolve  10  Gm.  peptone  (Witte's)  with  gentle  heating  in  200  to  300 
cc.  of  water. 

2.  Dissolve  0.5  Gm.  trypsin  in  20  cc.  of  water  by  shaking  and  gentle  heating 
(less  than  4o°C.). 

3.  Mix  and  digest  for  2  to  3  hours  at  38°C.,  stirring  gently  every  15  minutes. 

4.  Adjust  reaction  to  neutral,  add  water  to  make  1000  cc.,  boil,  filter,  tube 
and  sterilize  in  an  autoclave. 

This  medium  is  superior  to  Dunham's  for  detection  of  indol  production. 

Ox-bile  Bouillon. 

1.  Plain  bouillon,  100  c.c. 

2.  Ox  bile,  100  cc. 

Sterilize  in  steam  sterilizer  20  minutes  each  day  for  3  days, 

Bile  Medium  (Buxton  and  Coleman's). 

1.  Ox  bile,  900  cc. 

2.  Glycerin,  100  cc. 

3.  Peptone,  2  Gm. 

Sterilize  in  steam  sterilizer  20  minutes  each  day  for  3  days. 

Hiss  Serum  Water  Medium. 

1.  Beef-blood  serum,  100  cc. 

2.  Distilled  water,  300  cc. 

3.  Glucose  or  other  sugar,  4  Gm. 

4.  Sat.  aqueous  sol,  litmus  q.  s.  to  make  blue. 

Sterilize  in  steam  sterilizer  20  minutes  each  day  for  3  days 

MacConkey's  Bile  Salt  Medium. 

1.  Sodium  taurocholate,  0.5  Gm. 

2.  Peptone,  2  Gm. 

3.  Water,  100  cc. 


CULTURE    MEDIA  43 

Steam  for  2  hours,  filter  while  hot,  place  in  ice  box  over  night;  filter,  add  0.25 
cc.  of  i  per  cent,  aqueous  sol.  neutral  red  (freshly  prepared)  and  sugar  J£  or 
i  per  cent.  Glucose,  dulcite  or  adonite  J^  per  cent. — other  sugars  i  per  cent. 
Sterilize  in  steam  sterilizer  10  minutes  each  day  for  2  days. 

Gelatin  Medium. 

1.  Plain  bouillon,  100  cc. 

2.  Gelatin  (best  French  leaf),  10  to  30  Gm. . 

Break  up  gelatin,  allow  it  to  soak  in  the  bouillon  for  5  minutes;  then,  heat 
and  stir  until  dissolved  and  adjust  reaction. 

Sterilize  in  steam  sterilizer  for  15  minutes  each  day  for  3  days. 

Modifications  of  gelatin,  such  as  glucose,  lactose,  saccharose,  or  other  addi- 
tions and  coloring  with  litmus,  are  made  by  adding  from  i  to  2  per  cent,  of  the 
sugar  required  and  tinting  is  accomplished  by  adding  sufficient  sat.  aqueous 
solution  of  litmus  to  give  a  faint  blue  tint. 

Agar  Medium. 

1.  Plain  bouillon,  100  cc. 

2.  Agar,  i  to  3  Gm. 

Either  shredded  or  powdered  agar  may  be  used,  but  for  most  purposes  the 
former  is  preferable.  If  shredded  agar  is  used  it  should  be  cut  into  small  pieces 
before  putting  it  into  the  bouillon. 

Boil  and  stir  until  agar  is  dissolved,  add  water  to  make  up  for  the  loss  by 
evaporation,  adjust  reaction  and  filter  in  a  warm  place,  as  agar  solidifies  at 
about  35°C. 

Sterilize  in  autoclave. 

When  sugars  alone,  or  sugars  and  litmus,  are  added  to  agar,  it  is  done  the 
same  as  directed  for  bouillon  and  gelatin,  and  sterilized  15  minutes  each  day 
for  3  days. 

Glycerin  Agar  Medium.- — Prepare  agar  as  directed;  then,  add  6  per  cent 
glycerin. 

Sterilize  in  autoclave. 

Russel's  Double  Sugar  Agar  Medium. 

1.  Plain  agar  (3  per  cent.),  100  cc. 

2.  Lactose,  i  Gm. 

3.  Glucose,  o.i  Gm. 

Sat.  aqueous  sol.  litmus  violet  tint. 

Sterilize  in  tubes  in  steam  sterilizer  15  minutes  each  day  for  2  days  and  slant. 

Endo's  Medium. 

1.  Nutrient  agar  (3  per  cent.),  1000  cc. 

2.  Lactose,  10  Gm. 

3.  Filtered  sat.  sol.  basic  fuchsin  in  95  per  cent,  alcohol,  3  cc. 

4.  Sodium  sulphite  solution  (10  per  cent.),  30  cc. 

Liquefy  agar  in  Arnold  steam  sterilizer.     Dissolve  in  it  the  lactose.     Test 


44         «  MEDICAL  BACTERIOLOGY 

reaction.  Best  results  are  obtained  where  the  medium  is  neutral  or  just  slightly 
alkaline  to  litmus  paper.  Add  exactly  3  cc.  fuchsin  solution  and  30  cc.  sodium 
sulphite  solution  (or,  the  sod.  sulph.  being  hygroscopic,  you  may  add  instead  of 
30  cc.  of  the  10  per  cent,  sol.,  3  cc.  of  sat.  liquid  usually  found  in  even  tightly 
stoppered  bottles). 

The  resulting  mixture  is  quite  red  when  hot  but  loses  its  color  on  cooling. 
After  tubingy  sterilize  for  20  minutes  each  day,  for  3  days,  in  Arnold  steam 
sterilizer. 

Loeffler's  Malachite -green  Agar  Medium. 

1.  Meat  infusion  bouillon,  500  cc. 

2.  Distilled  water,  500  cc. 

3.  Neutralize. 

4.  Normal  hydrochloric  acid  sol.,  7.5  cc. 

5.  Shredded  agar,  30  Gm.  - 

6.  Boil  until  agar  dissolves,  then  neutralize  with  normal  sodium  hydrate. 

7.  Add  normal  sodium  carbonate  sol.,  5  cc. 

8.  Place  in  steam  sterilizer  for  2  hours;  then,  add — 

9.  10  per  cent,  aqueous  sol.  nutrose,  100  cc. 

10.  Sterilize  in  steam  sterilizer  15  minutes  each  day  for  2  days. 
When  ready  to  use,  liquefy  and  add  between  2  cc.  and  3  cc.  of  a  2  per  cent, 
aqueous  sol.  of  malachite  green  (Hochst  120)  to  each  10  cc.  of  medium. 

Conradi  and  Drigalski's  Medium. 

Minced  beef,  750  Gm. 

Water,  1000  cc. 

Put  in  shallow  dish,  in  ice  box,  over  night.     Skim  off  fat,  boil  for  i  hour, 
filter,  add  water  to  bring  volume  to  1000  cc. 
Add— 

Peptone  (Witte's),         10  Gm. 

Nutrose.  10  Gm. 

Calcium  chloride,  5  Gm. — 

boil;  add  30  Gm.  of  agar,  boil  until  agar  is  dissolved;  make  faintly  alkaline  to 
litmus  paper;  autoclave  for  i  hour;  filter  through  paper  and  autoclave  for  Y± 
hour. 
Add— 

Litmus  solution  (Kahlbaum) 150  cc. 

Lactose ; 15  Gm. 

Sterilize  in  Arnold  steam  sterilizer  for  Y±  hour. 
Add- 
Hot,  sterile,  o.i  per  cent,  solution  of  crystal  violet  (B.  Hochst),  10  cc. 

This  medium  favors  growth  of  the  typhoid  bacillus  and  inhibits  the  colon. 
On  it  typhoid  colonies  are  transparent,  blue-white,  and  cause  no  change  in 
color  of  medium.  Colon  colonies  are  red  and  opaque  and  the  medium  imme- 
diately surrounding  them  is  turned  from  blue  to  red. 


CULTUEE   MEDIA  45 

Blood-smeared  Agar  Medium.— Make  plain  agar,  put  in  tubes,  sterilize  in 
autoclave  and  slant.  When  the  agar  solidifies,  wash  a  finger  with  soap  and  hot 
water,  alcohol,  i:  1000  bichloride  sol.,  sterile  water,  then  dry  with  sterile  towel. 
Prick  the  finger  with  a  sterile  needle,  catch  the  blood  in  a  sterile  platinum  loop 
and  smear  it  over  the  entire  surface  of  the  agar. 

Blood  Agar  Medium. — Etherize  a  rabbit,  shave  and  asepticize  its  chest,  take 
a  lo-cc.  or  20-cc.  sterile  syringe  and  needle  and  withdraw  blood  from  heart, 
empty  blood  into  a  sterile  bottle  containing  sterile  glass  beads  and  shake  vigor- 
ously until  defibrinated.  Bring  temperature  to  45°C.  in  water  bath.  Have 
tubes  containing  10  cc.  sterile,  plain  agar,  liquefied,  and  at  a  temperature  of 
45°C.  With  a  sterile  pipette  add  5  cc.  of  blood  to  each  tube  of  agar,  mix  blood 
and  agar  and  slant. 

Blood  Alkali  Agar  Medium. — Obtain  ox  blood  in  slaughter  house.  Collect 
in  small  sterile  bottles  containing  glass  beads  (100  cc.  to  300  cc.,  cap  bottles), 
shake  until  defibrinated. 

Mix  equal  volumes  of  blood  and  normal  sodium  hydrate  solution.  Sterilize 
in  steam  sterilizer  15  minutes  each  day  for  2  days. 

Add  i  part  of  this  alkaline  blood  to  7  parts  sterile  plain  agar,  place  in  tubes 
and  slant.  Do  not  have  cotton  plugs  any  tighter  than  necessary. 

Loeffler's  Blood-serum  Medium/ — Blood  serum  (calf's  or  lamb's),  3  parts. 

Glucose  bouillon,  i  part. 

Put  in  tubes,  place  in  serum  sterilizer.  Heat  at  57°C.  i  hour  each  day  for 
6  days,  then  at  7o°C.  i  hour  each  day  for  2  days.  Or  heat  in  Arnold  steam 
sterilizer  at  8o°C.,  i  hour  each  day  for  3  days. 

A  Substitute  for  Ordinary  Blood  Serum.1- — "  Add  from  10  to  15  cc.  of  i  per 
cent,  glucose  bouillon  to  the  white  and  yolk  of  one  egg,  make  smooth  mixture 
in  a  mortar  and  place  in  tube.  Inspissate  and  sterilize  as  for  ordinary  serum 
slants." 

"When  this  medium  is  to  be  used  for  culturing  tubercle  bacilli,  add  about 
i  cc.  of  glycerin  bouillon  to  each  tube  before  final  sterilization  in  the  autoclave. 
The  cotton  plugs  should  be  paraffined  to  prevent  drying  of  the  slants  in  the 
incubator." 

Egg  Medium. — Select  a  clean,  fresh  egg  (not  more  than  24  hours  old)  that 
has  a  perfect  shell,  no  cracks;  shake  to  mix  white  and  yolk,  wash  with  warm 
sterile  water,  then  with  warm  i :  500  bichloride  solution  and  again  with  sterile 
water,  wipe  dry.  To  inoculate,  pierce  shell  with  sterile  needle  and  introduce 
bacteria  with  platinum  loop,  seal  opening  with  wax.  If  anaerobic  culture  is 
desired,  coat  egg  with  paraffin. 

Dorset's  Egg  Medium. — Selected  clean  fresh  eggs  with  perfect  shells.  Wash 
with  sterile  water,  then  with  5  per  cent,  phenol  and  finally  with  sterile  water. 
Break  with  a  sterile  knife  and  empty  the  eggs  into  a  sterile  flask. 

1  Stitt,  "Practical  Bacteriology,"  3d  edition,  p.  25. 


46  MEDICAL  BACTERIOLOGY 

Add  10  per  cent,  (by  weight)  of  sterile  distilled  water.  Mix.  Filter  through 
gauze,  tube,  slant  and  sterilize  in  steam  sterilizer  at  7o°C.  for  2%  hours. 

In  emptying  eggs  from  shell  care  must  be  taken  to  prevent  the  eggs  from 
contact  with  the  outside  of  the  shell  or  fingers;  when  mixing  with  water  and  tub- 
ing, bubbling  must  be  avoided. 

Potato  Medium. — Take  a  smooth,  new  potato,  wash  in  water  (hot),  hot  i :  500 
bichloride;  then,  in  several  changes  of  hot  water.  Cut  cylindrical  pieces  about 
i><2  inches  long  and  about  the  same  diameter  as  the  tubes  in  which  they  are 
to  be  placed.  Split  each  cylinder  diagonally  in  two  through  long  axis,  place 
each  piece  in  a  tube  with  the  thick  end  at  the  bottom  and  add  about  J^  inch  of 
distilled  water. 

Sterilize  in  autoclave. 

Petroff's  Medium. 

2  parts  egg  (whites  and  yolks). 

i  part  meat  juice. 

Gentian  violet  in  i :  10,000  dilution. 

Meat  juice — infuse  500  Gm.  of  veal  in  500  cc.  of  15  per  cent.  sol.  glycerin 
in  H2O  for  24  hours  and  strain  off  juice. 

Eggs — sterilize  shells  with  20  per  cent,  carbolic  acid  and  break  into  sterile 
beaker  and  mix  well.  Then  filter  through  gauze,  adding  i  part  of  the  infusion 
by  volume. 

Gentian  violet — add  sufficient  i  per  cent,  alcoholic  gentian  violet  to  make  a 
dilution  of  i :  10,000. 

Tube  and  sterilize  in  steam  sterilizer  or  inspissate  for  3  successive  days, 
8o°C.  for  i  hour  each  day. 

Modification  of  Petroff's  Medium. — (By  Williams-Burdick). 

1.  Egg-white   Solution. — This  is  made  by  diluting   the   egg  white   with 
10  parts  of  distilled  water  and  thoroughly  shaking.     The  fluid  is  opalescent 
and  contains  numerous  whitish  flakes.     To  clear  it,  pass  it  through  a  thin  layer 
of  cotton  and  then  heat  to  ioo°C.  to  hasten  precipitation.     It  is  then  filtered 
through  paper. 

2.  Egg-yolk  Solution. — The  yolks  are  diluted  with  10  parts  of  water  and 
well  stirred.     The  cloudy  emulsion  is  clarified  by  adding  normal  sodium  hydrox- 
ide.    Too  much  hydroxide  is  harmful  and  therefore  complete  solution  of  the 
yolk  is  not  desirable.     The  emulsion  should  be  slightly  turbid.     To  attain  the 
proper  degree  of  turbidity,  i  cc.  of  normal  NaOH  is  usually  added  to  each  100 
cc.  of  emulsion.     This  is  not  a  constant  amount,  however,  because  some  yolks 
will  be  completely  dissolved  by  less  than  half  this  amount  of  alkali.     The  solu- 
tion is  heated  to  ioo°C.  and  filtered. 

3.  Meat  Infusion. — 500  Gm.  of  finely  chopped  lean  veal  are  covered  with  i 
liter  of  water  containing  15  per  cent,  glycerin,  allowed  to  infuse  for  24  hours  and 
filtered;  5  Gm.  NaCl  are  added  and  the  infusion  heated  to  boiling.     It  is  again 


CULTURE    MEDIA  47 

filtered  and  then  rendered  plus  i  per  cent,  alkaline.     With  the  above  solutions, 
the  medium  is  made  as  follows: 

.Place  300  cc.  of  the  10  per  cent,  egg-white  solution  in  a  liter  flask;  300  cc. 
of  the  10  per  cent,  egg-yolk  solution  in  another  flask,  and  400  cc.  of  the  meat 
infusion,  to  which  is  added  15  Gm.  of  powdered  agar-agar,  in  a  third  flask. 
These  are  then  sterilized  in  the  autoclave  at  15  pounds  pressure  for  15  minutes. 
They  are  removed  from  the  sterilizer  and,  while  hot,  i  cc.  of  a  i  per  cent,  alco- 
holic solution  of  gentian  violet  is  added  to  the  broth-agar.  The  contents  of  this 
flask  are  now  poured  into  that  containing  the  egg  white  and  then  the  egg  yolk 
is  added.  The  whole  is  poured  back  and  forth  from  this  flask  to  another  so  as  to 
insure  thorough  mixing,  and  then  it  is  tubed  and  slanted.  The  tubes  are  left 
in  slanted  position  for  about  72  hours,  at  room  temperature,  until  the  contents 
are  well  set,  and  the  tubes  sealed  with  corks  or  paraffin. 

REACTION  OF  CULTURE  MEDIA 

The  most  common  organisms  which  are  both  saprophytic  and  pathogenic 
flourish  on  media  either  slightly  acid,  neutral  or  slightly  alkaline  in  reaction. 
Many  of  these  grow  most  luxuriantly  in  a  medium  having  a  reaction  indicated 
as  slightly  alkaline  when  tested  with  litmus  paper  (turn  red  paper  slightly  blue, 
do  not  turn  blue  paper  red).  For  general  purposes,  media  tested  with  litmus 
paper  and  adjusted  to  give  a  slightly  alkaline  reaction,  do  well. 

Plain  bouillon,  agar  and  gelatin  media,  when  made,  are  usually  acid  to  litmus 
paper.  When  it  is  necessary  to  add  acid  or  alkali,  solutions  of  hydrochloric  acid 
or  sodium  hydrate  are  used. 

Although  this  easy  method  of  testing  and  adjusting  the  reaction  of  culture 
media  is  ordinarily  satisfactory,  it  has  faults  that  make  more  accurate  methods 
preferable,  at  least  for  some  purposes.  The  observations  of  different  workers 
are  comparable  only  when  the  culture  media  they  use  is  uniform  in  reaction. 
This  is  especially  true  and  important  in  culturing  milk,  water,  sewage  and  food- 
stuffs to  determine  the  "number  of  bacteria  contained.  For  this  reason  the 
American  Public  Health  Association  has  recommended  the  following  method 
of  testing  and  adjusting  the  reaction  of  culture  media: 

Reagents — freshly  prepared  J/2  per  cent,  solution  of  phenolphthalein  in 
50  per  cent,  alcohol — indicator. 

Normal,  tenth  normal  and  twentieth  normal  solutions  of  hydrochloric  acid 
and  sodium  hydrate. 

Place  5  cc.  of  culture  medium  in  porcelain  dish,  add  45  cc.  distilled  water, 
boil  for  3  minutes,  add  i  cc.  of  phenolphthalein  (indicator);  if  acid;  the  contents 
of  the  dish  snow  no  change  of  color;  if  alkaline,  turn  red.  Usually  the  reac- 
tion is  acid;  in  this  case  tenth  normal  NaOH  is  added,  drop  by  drop,  from  a 
graduated  burette  until  the  neutral  point  is  reached;  the  amount  of  NaOH 
required  to  neutralize  is  recorded,  and  the  experiment  repeated,  using  twentieth 
normal  NaOH. 

Should  the  contents  of  the  dish  turn  red  when  the  phenolphthalein  is  added 
it  is  alkaline  and  titration  would  be  done  with  T0  and  ,.  HC1. 


48  MEDICAL  BACTERIOLOGY 

For  preparation  of  synthetic  media,  see  "Preliminary  Report  on  Synthetic 
Media,"  C.  J.  T.  Borland,  Journal  of  Bacteriology,  1916,  vol.  i,  No.  2,  p.  135. 

During  titration  the  contents  of  the  dish  should  be  constantly  stirred.  When 
NaOH  is  being  added,  as  each  drop  falls  from  the  burette  it  causes  a  bright  rose 
red  to  appear,  which  varnishes  on  stirring,  until  the  neutral  point  is  reached. 

When  the  contents  of  the  dish  show  a  faint,  but  distinct  pink  color,  that  does 
not  disappear  on  stirring  and  heating,  the  neutral  point  has  been  reached. 

Having  neutralized  a  measured  sample  of  culture  medium  with  a  measured 
amount  of  acid  or  alkali,  it  is  easy  to  compute  just  what  quantity  of  one  or  the 
other  need  be  added  to  the  bulk  of  medium  to  produce  any  desired  degree  of 
acidity  or  alkalinity — e.g.,  if  we  are  using  a  —  NaOH  solution  and  find  that  the 
neutral  point  has  been  reached  when  2  cc.  of  it  have  been  dropped  into  the  dish 
containing  5  cc.  of  the  culture  medium,  then  to  neutralize  100  cc.  of  the  culture 
medium,  twenty  times  as  much  -0  NaOH  would  be  required,  or  40  cc.  of  I0 
NaOH.  As  i  cc.  of  -x  NaOH  is  equivalent  to  10  cc.  of  10  NaOH,  then  4  cc. 
Y  NaOH  would  neutralize  100  c.c  of  the  culture  medium. 

In  establishing  a  standard  for  this  method  of  titration  the  plus  sign  is  taken 
to  indicate  acidity,  the  minus  sign  to  indicate  alkalinity 

Culture  media  having  a  reaction  that  would  require  the  addition  of  i  cc. 
of  normal  sodium  hydrate  to  each  100  cc.  of  media  to  neutralize  it  is  said  to  have 
a  reaction  of  +i.  In  other  words,  +i  indicates  that  the  media  is  so  acid  that 
i  per  cent,  of  normal  sodium  hydrate  would  be  required  to  neutralize  it. 

For  general  purposes  and  when  the  number  of  bacteria  per  cubic  centimeter 
is  to  be  determined  in  milk,  foodstuffs,  water,  sewage,  etc.,  +1.5  is  the  standard 
reaction. 

As  a  matter  of  fact  when  standard  peptone,  sodium  chloride,  meat  extract 
or  meat  infusion  are  used  in  making  culture  media,  when  completed,  the  reac- 
tion nearly  always  falls  between  +0.5  and  +1.5.  If  the  reaction  is  found 
to  lie  between  these  limits  no  attempt  should  be  made  to  adjust  it.  If  it  falls 
outside  these  limits  it  should  be  adjusted  to  +1.5. 

The  growth  of  bacteria  is  largely  influenced  by  variations  in  the  titratable 
acidity  of  culture  media.  Some  organisms  are  much  more  sensitive  to  varia- 
tions in  this  than  others,  and  bacteriologists  since  the  days  of  Pasteur  have  given 
careful  attention  to  this  fact.  Recent  investigations  have  shown  that  in  some 
instances,  probably  in  many,  the  hydrogen  ion  concentration  is  equally  as  im- 
portant as  the  titratable  acidity  of  media.  At  the  present  time  no  method  of 
measuring  or  adjusting  this  is  in  general  use.  Undoubtedly,  in  the  near  future 
this  will  be  done.  The  student  is  recommended  to  read  Clark  and  Lubs  mono- 
graph on  this  subject,  which  is  the  clearest  and  most  informative  presentation 
of  the  subject.  "The  Colorimetric  Determination  of  Hydrogen  Ion  Concen- 
tration and  Its  Applications  in  Bacteriology,"  Journal  of  Bacteriology,  vol.  ii, 
No.  I,  January,  1917. 

FILTRATION  OF  CULTURE  MEDIA 

Culture  media  must  be  clear.     This  is  usually  accomplished  by  filtration. 


CULTURE   MEDIA  49 

Bouillon  needs  to  be  passed  through  several  thicknesses  of  filter-paper,  some- 
times repeatedly,  while  it  is  hot  and  again  when  cool.  Gelatin  and  agar  are 
cleared  by  one  passage  through  a  single  thickness  of  filter-paper.  Filter-papers 
should  be  moistened  with  water  and  allowed  to  stand  a  few  minutes  before  media 
is  poured  on  them. 

If  the  white  of  egg  is  added  and  the  media  then  boiled  for  5  minutes  before 
filtration,  much  of  the  suspended  matter  is  caught  in  the  coagulum  that  forms 
and  less  filtration  is  required.  At  least  one  egg  to  every  1000  cc.  of  media 
should  be  used,  the  whites  well  beaten  and  then  thoroughly  mixed  with  the 
media  by  stirring. 

When  egg  has  been  added  gelatin  and  agar  can  be  cleared  by  filtering  through 
cotton,  provided  it  is  done  carefully.  A  piece  of  gauze  should  be  placed  in  a 
funnel,  upon  this  a  piece  of  cotton  about  %  inch  thick,  with  all  the  fibers 
running  in  one  direction,  should  be  placed.  Upon  this  place  a  second  piece  of 
cotton  the  same  thickness,  with  its  fibers  at  right  angles  to  those  of  the  bottom 
layer.  Moisten  with  water  and  smooth  the  edges  to  the  side  of  the  funnel; 
then,  gradually  run  culture  media  upon  it  so  as  to  prevent  the  media  from 
escaping  between  the  cotton  and  side  of  funnel.  Filtration  through  cotton  is 
quicker  than  through  paper. 

Blood  serum  to  be  used  in  the  fluid  state  and  other  media  that  cannot  be 
heated  are  both  cleared  and  sterilized  by  filtration  through  unglazed  porcelain 
tubes. 

CONTAINERS  FOR  CULTURE  MEDIA 

Media  kept  in  bulk  are  placed  in  flasks  of  from  100  cc.  to  1000  cc.  capacity; 
otherwise  they  are  kept  in  test-tubes.  These  containers  should  be  made  of 
alkali-free  glass,  be  clean  and  clear.  They  are  plugged  with  cotton  and  steril- 
ized in  a  hot-air  sterilizer  before  use.  The  plugs  are  made  of  clean  non-absorbent 
cotton.  A  smooth  piece  of  cotton  is  placed  across  the  mouth  of  a  tube  and 
pushed  in  with  a  pencil  or  similar  instrument;  the  resultant  plug  will  be  tight 
or  loose,  according  to  the  size  of  the  piece  of  cotton,  and  this  must  be  regulated 
so  that  the  plug,  extending  into  the  tube  from  J^  to  i  inch,  will  be  firm  enough 
to  permit  lifting  of  the  tube  by  grasping  the  cotton  and  still  not  so  tight  that  it 
cannot  be  replaced  with  ease  when  removed.  The  cotton  plug  is  made  to  extend 
about  J^  inch  above  the  top  of  the  tube  so  that  it  can  be  readily  removed  and 
held  without  touching  the  portion  that  enters  the  tube. 

Media  in  cotton-plugged  containers  tend  to  dry  out,  the  smaller  the  bulk, 
the  more  rapidly  does  it  dry  out. 

When  necessary  this  is  guarded  against  by  placing  rubber  caps  over  the  top 
of  the  tube  and  plug  or  by  dipping  the  top  of  the  tube  and  plug  in  melted 
paraffin. 

Culture  media  keep  best  when  placed  in  a  clean  refrigerator. 

Should  the  portion  of  a  tube  that  the  plug  comes  into  contact  with  be  wet 
with  culture  media,. more  or  less  cotton  adheres  .when  the  plug  is  removed,  in- 
terfering with  the  introduction  and  removal  of  bacteria.  This  is  avoided  by 


MEDICAL  BACTERIOLOGY 


filling  the  tubes  with  a  jacketed  test-tube  filler  constructed  to  guard  the  upper 
part  of  the  tube  from  contact  with  media. 

After  media  has  been  run  into  tubes,  they  are  never  tilted,  slanted  or  shaken 
to  a  degree  that  would  send  the  media  into  contact  with  the  plugs. 

When  using  tubes  5  or  6  inches  long  they  are  usually  filled  to  a  depth  of 


FIG.  7. 


SMEAR-CULTURE.     This    tube    shows    the 
rubber  cap  used  to  prevent  drying. 


STAB-CULTURE.     A  rubber  stopper  may  be 
used  to  prevent  drying. 


about  2  inches  with  liquid  media,  such  as  bouillon,  about  the  same  with  solid 
media  intended  for  plating  and  stab  cultures. 

To  obtain  surface  growth  on  solid  media  in  test-tubes,  immediately]  after 
sterilization,  while  the  media  is  still  liquid,  the  tubes  are  slanted  and  permitted 


FIG.  8. — PETRI  DISH   (MacNeal). 

to  remain  so  until  the  media  solidifies;  these  tubes  should  contain  such  a  quantity 
of  media  and  be  slanted  at  sucK  an  angle  that  when  solid  the  media  will  entirely 
fill  the  bottom  %  inch  of  the  tube,  the  upper  portion  being  at  least  an  inch  below 
the  plug. 

Covered  glass  dishes — Petri  dishes — are  frequently  employed  when  a  broad 
surface  growth  on  solid  media  is  desired.     They  are  shallow  and  intended   to 


CULTURE    MEDIA  51 

contain  a  comparatively  thin  layer  of  culture  media,  so  that  media  becomes  dry 
in  them  after  several  days.  Consequently,  Petri  dishes  only  have  media  placed 
in  them  at  the  time  one  intends  to  plant  bacteria. 

The  media  intended  for  use  in  dishes  is  stored  in  flasks  or  tubes.  When  a 
plate  is  to  be  planted,  a  flask  or  tube  of  sterile  media  is  liquefied  in  a  water  bath 
and  poured  into  a  sterile  dish;  when  solid  the  substance  to  be  planted  is  dropped 
on  the  surface  or  streaked  over  it  with  a  sterile  platinum  wire  or  glass  rod. 
Another  method  is  to  liquefy  a  tube  of  culture  media,  cool  it  to  4o°C.,  drop  the 
substance  to  be  cultured  into  it,  mix  by  turning  the  tube  upside  down  several 
times,  and  then  pour  the  contents  of  the  tube  into  a  Petri  dish.  As  a  precau- 
tion against  contamination  the  cover  of  a  Petri  dish  is  lifted  as  little  as  possible 
and  media  is  not  allowed  to  run  over  the  edges.  Dishes  are  not  moved  nor 
placed  in  an  incubator  until  the  contents  have  solidified. 

CULTURE  TECHNIQUE 

Liquids  containing  bacteria  that  are  to  be  cultured  are  transferred  to  tubes 
of  culture  media  with  sterile  glass  pipettes  or  with  loops  of  platinum  wire;  solidi 
and  macroscopic  masses  of  bacteria  are  commonly  handled  with  loops  of  plats 
num  wire  except  when  stab  cultures  are  to  be  made,  then  a  straight  piece  of 
wire  or  a  needle  is  used. 

When  a  tube  of  culture  media  is  to  be  planted  it  is  desirable  to  have  the  air 
as  still  and  as  free  of  dust  as  possible,  windows  and  doors  are  closed  and  one 
avoids  breathing  directly  upon  or  over  exposed  surfaces  of  media.  The  cotton 
plugs  are  removed  from  flasks  or  tubes  containing  culture  media  by  grasping  the 
plug  between  the  little  finger  and  palm  of  the  hand,  or  between  the  other  fingers, 
care  being  taken  to  avoid  touching  the  inside  of  the  tube.  While  the  plug  is 
held  between  the  fingers,  that  part  which  enters  the  tube  must  not  come  into 
contact  with  anything.  Just  before  replacing  the  plug  it  is  well  to  pass  it 
through  a  flame  to  burn  off  any  organisms  that  may  have  settled  on  it.  The  less 
frequently  tubes  are  opened  and  the  shorter  the  time  the  plug  is  out  of  the  tube, 
the  less  danger  of  contamination.  After  removing  the  plug,  the  open  end  of  the 
tube  is  flamed  before  introducing  or  removing  bacteria;  it  is  again  flamed  before 
replacing  the  plug. 

If  a  pipette  is  used  to  inoculate  media  its  contents  are  dropped  into  or  upon 
the  media  without  bringing  the  pipette  into  contact  with  the  media  or  the  upper 
portion  of  the  tube.  When  inoculating  liquid  media  with  platinum  loop,  the 
loop  is  held  so  that  it  does  not  touch  the  side  of  the  tube  until  submerged  in  the 
media,  then  it  is  given  a  quick  turn  and  removed  with  the  same  care  as  when 
inserted. 

Material  containing  bacteria  to  be  cultivated  on  the  surface  of  solid  media 
(either  slants  in  tubes  or  plates)  must  be  gently  dropped,  smeared  or  streaked 
across  the  surface  to  avoid  breaking  the  smooth  surface. 

To  determine  the  character  of  growth  beneath  the  surface,  to  observe  gas 
formation  and  other  phenomena,  what  are  known  as  shake  cultures  are  sometimes 
made  in  solid  media^  usually  in  gelatin.  A  tube  of  media  is  liquefied  and  cooled 


52  MEDICAL  BACTERIOLOGY 

to  4o°C.,  the  bacteria  are  put  in  it  and  the  tube  shaken  to  distribute  them,  it  is 
then  solidified. 

Stab  cultures  made  in  solid  media,  usually  gelatin  or  agar,  are  made  by 
thrusting  a  needle  or  straight  platinum  wire  coated  with  bacteria  through  the 
long  axis  of  a  tube  of  media  nearly  to  the  bottom. 

Gas  formation  by  bacteria  is  best  observed  by  cultivating  in  liquid  media  in 
fermentation  tubes,  tubes  so  constructed  that  displacement  of  fluid  by  generated 
gas  is  easily  discernible. 

The  best  form  of  fermentation  tube  consists  of  an  upright  cylindrical  tube, 
about  5  inches  long  and  ^g  inch  in  diameter,  closed  at  the  top  and  connected 
at  the  bottom  by  a  narrower  short  U-tube  to  a  spherical  chamber,  of  about  10 
cc.  capacity,  which  has  an  opening  at  the  top.  These  fermentation  tubes  are 
plugged  with  cotton  and  sterilized  in  a  hot-air  oven.  The  media  to  be  used  in 
them  is  best  handled  if  tubed,  sterilized  and  stored  in  test-tubes;  each  test-tube 
containing  sufficient  medium  to  completely  fill  the  upright  arm  and  one-half 
of  the  spherical  portion  of  a  fermentation  tube. 

When  a  test  is  to  be  made  the  suspected  material  is  first  placed  in  the  fer- 
mentation tube  and  the  culture  medium  then  poured  into  the  bulb.  By  tipping 
the  fermentation  tube  the  suspected  substance  and  culture  medium  mix  and 
flow  into  the  upright  arm.  Every  bubble  of  air  must  be  displaced  from  the 
upright  portion  of  the  fermentation  tube,  so  that  the  formation  of  gas  in  the 
tube  may  be  recognized,  as  it  accumulates  at  the  top  of  the  upright  arm. 

Indol. — Some  bacteria  produce  indol  (C8H7N)  when  cultured  on  appropriate 
media,  and  this  fact  is  taken  advantage  of  in  the  identification  and  differentia- 
tion of  certain  organisms.  It  must  be  remembered  that  an  organism,  like  the 
colon  bacillus,  which  ordinarily  produces  indol,  may  at  times  temporarily  lose 
its  ability  to  do  so. 

Dunham's  solution  has  been  largely  used  as  a  culture  medium  favorable  to 
indol  production,  but  the  Trypsinized  peptone-water  of  Rivas  seems  superior. 

The  organism  to  be  tested  is  planted  in  a  test-tube  containing  Dunham's 
or  Rivas'  medium  and  incubated  at  37°C.  If  only  one  tube  is  planted  it  is 
tested  for  indol  after  48  hours  of  incubation.  Sometimes  sufficient  indol  is 
produced  in  6,  12  or  24  hours  to  be  detected  and  hence  when  the  determination 
is  desired  as  early  as  possible,  it  is  expedient  to  plant  several  tubes  and  examine 
one  6  hours,  another  12  hours,  another  24  hours,  and,  finally,  one  48  hours  after 
incubation. 

The  presence  of  indol  is  disclosed  by  adding  5  to  20  drops  of  a  0.2  per  cent, 
solution  of  potassium  nitrite,  shake  the  tube,  then  add  slowly  an  equal  quantity 
of  a  25  per  cent,  solution  of  pure  sulphuric  or  hydrochloric  acid.  If  indol  is 
present  the  culture  medium  turns  red,  immediately  after  the  addition  of  acid, 
or  several  minutes  later. 

For  an  elaborate  discussion  of  indol  production  by  bacteria,  see  "Studies 
on  Indol,"  Rivas,  D.,  Centralblatt  fur  Bakteriologie,  Parasitekunde  und 
Infektionskrankheiten,  Orig.,  1912,  Ixiii  Bd. 

Plating. — The  cultivation  of  bacteria  in  broad,  shallow,  circular,  covered 


CULTURE    MEDIA  53 

glass  dishes  (Petri  dishes)  is  referred  to  as  plating.  The  advantages  of  this 
method  are  the  possibility  of  procuring  discrete  colonies  when  quantities  are 
planted  that  would  give  a  confluent  growth  in  tubes,  greater  facility  of  study- 
ing the  appearance  of  individual  colonies  as  they  occur  on  the  medium,  greater 
possibility  of  removing  a  single  colony  from  the  medium  with  a  platinum  loop 
without  contaminating  it  by  contact  with  others.  Therefore,  when  substances 
contain  several  species  of  organisms  and  it  is  desirable  to  isolate  one  or  more  of 
them,  plating  is  usually  the  most  convenient  method;  also,  when  it  is  desirable 
to  determine  the  number  of  bacteria  per  cubic  centimeter  in  a  substance,  plating 
usually  affords  the  best  means  of  so  doing. 

Most  bacteria  associated  with  disease  grow  best  or  only  grow  at  or  near  the 
temperature  of  the  human  body,  hence  cultures  are  usually  placed  in  an  incu- 
bator at  37°C.  to  facilitate  bacterial  growth.  Saprophytic  bacteria  are  usually 
incubated  at  room  temperature  or  at  2O°C.  or  25°C. 

Aerobic  bacteria,  those  that  require  oxygen  to  develop,  grow  well  in  bouillon 
and  on  the  surface  of  solid  media  in  tubes  or  flasks  stoppered  with  cotton  plugs. 
Sufficient  oxygen  percolates  through  the  cotton  when  the  tubes  are  kept  in  a 
room,  closet  or  incubator  where  the  atmosphere  is  air. 

Anaerobic  bacteria,  those  that  grow  only  in  the  absence  of  free  oxygen,  are 
usually  planted  in  media  containing  i  to  2  per  cent,  of  glucose,  because  glucose 
is  a  reducing  agent  and  free  oxygen  does  not  exist  in  media  containing  it. 

When  better  means  are  not  available  anaerobes  can  be  cultivated  by  taking 
tubes  half -full  of  solid  glucose  agar,  making  deep  stab  inoculations  in  the  center 
of  the  media,  and  covering  the  surface  with  an  inch  or  more  of  sterile  liquid 
petrolatum. 

Anaerobic  cultures  are  usually  made  by  placing  the  inoculated  tubes  or  Petri 
dishes  in  air-tight  jars,  from  which  the  oxygen  is  removed  either  by  extraction, 
displacement  or  absorption,  or  a  combination  of  these  methods.  For  extrac- 
tion and  displacement  specially  devised  jars,  fitted  with  taps  and  stop-cocks 
are  required.  Exhaustion  is  achieved  by  pumping  out  the  air  and  leaving  a 
vacuum.  Displacement  is  achieved  by  introducing  hydrogen  through  one  tap 
while  the  other  is  open  so  that  air  and  oxygen  pass  out  as  hydrogen  enters. 
When  the  atmosphere  is  entirely  devoid  of  oxygen  the  stop-cocks  are  closed. 
Hydrogen  is  generated  for  this  purpose  in  a  Kipp's  apparatus,  from  sulphuric 
acid  and  zinc.  It  is  passed,  in  the  order  mentioned,  through  three  wash  bottles; 
the  first  containing  10  per  cent,  lead  acetate  solution,  the  second  10  per  cent, 
silver  nitrate  solution  and  the  third  a  10  per  cent,  solution  of  pyrogallic  acid  in 
10  per  cent,  sodium  hydrate  solution.  It  is  then  delivered  into  the  jar  contain- 
ing culture  tubes  or  plates. 

Absorption  of  oxygen  does  not  require  special  apparatus,  any  jar  having  a 
ground-glass  stopper  or  screw  cap  that  can  be  made  air-tight  will  do.  The  tubes 
or  plates  are  placed  in  the  jar,  on  a  support  that  elevates  them  an  inch  or  more 
above  the  bottom.  About  J^  inch  of  pyrogallic  acid  is  placed  in  the  bottom  of 
the  jar,  potassium  hydrate  solution  is  poured  upon  it  and  the  jar  quickly  sealed. 
Potassium  pyrogallate  is  formed  and  it  absorbs  the  oxygen.  To  2  to  4  Gm. 


54 


MEDICAL  BACTERIOLOGY 


of  pyrogallic  acid  add  109  Gm.  of  potassium  hydrate  dissolved  in  145  cc.  of 
water.  Should  a  ground-glass-stoppered  jar  be  used,  the  ground  surface  should 
be  anointed  with  vaseline  to  insure  an  air-tight  joint,  and  for  the  same  reason 
washers  are  used  on  screw-top  jars. 

Quantity  of  material  to  be  planted  on  or  in  culture  media  to  obtain  a  growth 
is  governed  by  a  number  of  factors,  among  which  are  the  following: 

Some  organisms  grow  luxuriantly  when  planted  on  culture  media,  regardless 
of  whether  taken  directly  from  tissue  (sputum, 
pus,  blood,  urine,  etc.)  or  from  previous  laboratory 
cultures;  others  grow  luxuriantly  after  several 
transplants  from  culture  media  to  culture  media, 
but  show  very  scant  growth,  and  that  irregularly, 
when  first  transplanted  from  tissue  or  exudate 
to  culture  media;  some  never  grow  abundantly  and 
are  irregular  in  growing,  no  matter  how  often  they 
are  transplanted  from  media  to  media  and  some  grow 
abundantly  on  certain  media  and  poorly  on  other 
media.  For  these  reasons  the  quantity  of  any  sub- 
stance planted  on  media  to  obtain  a  culture  varies 
according  to  the  number  of  organisms  contained 
in  the  substance,  the  peculiarities  of  the  particular 
organism  to  be  cultured  and  the  medium  upon 
which  it  is  planted,  also  the  quantity  of  growth 
desired. 

To  determine  the  number  of  bacteria  per 
cubic  centimeter  contained  in  any  substance,  for 
the  isolation  of  a  single  organism  from  a  mixture 
and  for  future  cultural  and  biological  studies  of 
bacteria,  one  must  obtain  a  growth  in  which  the 
colonies  remain  discrete  and  far  enough  apart  to 
permit  the  removal  of  a  single  colony  without 
coming  into  contact  with  others — 20  to  200  on  a 
Petri  dish. 

When  dealing  with  a  substance  rich  in  bacteria  that  grow  abundantly— 
as  feces  containing  colon  bacilli — the  almost  invisible  amount  that  adheres  to 
a  small  platinum  loop  gently  touched  to  it,  is  sufficient  to  plant  one  or  several 
tubes  or  plates  and  produce  an  abundant  growth. 

Very  many  organisms,  which  ordinarily  grow  abundantly  on  numerous 
media,  at  times  fail  to  grow  at  all  when  transplanted;  such  failure  to  grow  is  a 
more  frequent  occurrence  when  organisms  difficult  to  cultivate  are  dealt  with 
and  hence  it  is  always  advisable  to  plant  two,  three  or  more  tubes  or  plates, 
not  one. 


FIG.  9. — ARRANGEMENT  OF 
TUBES  FOR  CULTIVATION  OF 
ANAEROBES  BY  BUCHNER'S 
METHOD  (MacNeal.) 


CHAPTER  VII 
STAPHYLOCOCCI 

Staphylococci  are  commonly  present  in  air,  dust  and  soil,  frequently  in  the 
mouth  and  intestinal  tract  of  healthy  as  well  as  diseased  people.  Almost 
constantly  upon  the  scalp,  hands  and  other  portions  of  the  body  surface,  in 
milk  and  foodstuffs. 

Many  saprophytic  non-pathogenic  and  saphrophytic  and  pathogenic  species 
of  Staphylococci  have  been  isolated  and  described;  most  of  the  latter  have  very 
rarely  been  found  associated  with  disease  in  man  and  of  these  some  appear  to 
have  a  selective  affinity  for  certain  organs  of  the  body,  similar  to  the  predilec- 
tion for  certain  organs,  according  to  Rosenow,  shown  by  streptococci. 


FIG.  10. — STAPHYLOCOCCUS,  PURE  CULTURE.     STAINED  WITH  METHYLENE  BLUE. 
(4  X  eyepiece  and  Ma  oil  immersion  objective.) 

Of  the  various  organisms  that  infect  man,  Staphylococci  are  among  the  most 
frequent  offenders,  and  the  vast  majority  of  such  infections  .are  caused  by  one 
or  other  of  the  following:  staphylococcus  albus,  staphylococcus  aureus  and 
staphylococcus  citreus.  Practically,  these  are  alike  in  all  respects,  except  pig- 
ment production.  Staphylococcus  albus  produces  a  white  pigment;  aureus,  a 
pigment  that  varies  from  light  golden  yellow  to  deep  brownish-yellow;  citreus, 
lemon  or  greenish-yellow  pigment.  Chromogenesis  appears  when  colonies 
develop  on  media  in  an  aerobic  atmosphere,  under  anaerobic  conditions  pigment 
is  not  produced.  Old  cultures  tend  to  lose  their  chromogenic  power. 

55 


56  MEDICAL  BACTERIOLOGY 

Morphology. — Staphylococci  are  spherical,  unicellular  organisms,  from  0.5 
to  i  .o  /x  in  diameter.  The  characteristic  of  their  arrangement  is  irregularity. 
They  occur  in  masses,  some  of  which  resemble  bunches  of  grapes;  irregularly 
arranged  groups  of  three,  four  and  five  are  numerous;  fragmentation  of  these 
irregular  masses  results  in  accidental  arrangement  of  a  few  in  pairs  or  short 
chains  as  well  as  singly. 

Motility. — In  hanging-drop  preparations  Brownian  movement  is  observed, 
but  not  true  motility;  Staphylococci  are  non-motile. 

Staining. — Staphylococci  stain  with  all  the  usual  anilin  dyes  and  are  Gram 
positive. 

Culture.— Staphylococci  are  aerobic  and  facultative  anaerobic.  They  grow 
at  any  temperature  between  io°C.  and  4b°C.,  most  luxuriantly  at  or  near  body 
temperature,  37°C.  All  the  ordinary  media  are  suitable  for  cultivating  Staphy- 
lococci. Media  that  are  slightly  alkaline  are  most  favorable,  but  they  grow 
well  if  the  reaction  is  neutral  or  slightly  acid. 

Blood  Serum.- — On  coagulated  blood  serum  growth  is  rapid,  at  first  trans- 
parent; then,  the  color  becomes  rather  white,  and  within  24  to  48  hours  chromo- 
genesis  appears.  The  colonies  are  round,  elevated,  smooth-edged  and  have  a 
moist,  glistening  appearance.  They  vary  in  size,  some  smaller  and  a  few  larger 
than  a  pin  head,  in  several  days  they  coalesce  and,  after  a  time,  slight  liquefac- 
tion of  the  media  is  produced  by  some  strains. 

Agar. — Growth  on  agar  slants  in  tubes  appears  the  same  as  on  serum, 
but  liquefaction  of  agar  never  occurs.  On  agar  plates  colonies  appear  as  al- 
ready described;  they  tend  to  remain  discrete;  coalescence  does  not  usually 
occur. 

Gelatin. — Surface  cultures  on  plates  and  slant  tubes,  incubated  at  room 
temperature  2o°C.  to  25°C.)  show  colonies  within  24  to  48  hours.  After  several 
days  the  media  adjacent  to  the  colonies  liquefies  so  that  each  colony  is  sur- 
rounded by  a  basin-like  zone  of  liquid  gelatin.  As  time  goes  on,  liquefaction 
progresses,  zones  of  liquefaction  merge  and  finally  the  entire  media  becomes 
fluid. 

Gelatin  Stab  Cultures. — First  show  granular  growth  along  the  stab;  several 
days  later  liquefaction  begins.  It  starts  at  the  surface  of  the  stab;  as  it  pro- 
gresses it  becomes  funnel-shaped,  the  greatest  area  of  liquefaction  being  at  the 
surface,  the  track  gradually  narrowing  toward  the  bottom.  A  cloudy  sediment, 
white  or  yellowish,  forms  in  the  liquefied  gelatin.* 

Bouillon. — Becomes  cloudy  in  12  to  24  hours;  then,  a  white  precipitate  falls 
to  the  bottom,  the  media  continuing  cloudy.  After  several  days  the  sediment 
becomes  yellowish  if  staphylococcus  aureus  or  citreus  is  present. 

Milk  is  acidulated  and  coagulated.  ' 

Potato  is  the  most  favorable  medium  for  pigment  production.     An  abundant 

*  Different  strains  of  Staphylococci  vary  in  their  effect  on  gelatin  nitrates  and  carbohydrates. 
Some  liquefy  gelatin  more  slowly  than  others,  some  do  not  liquefy  it  at  all.  Some  act  on  all  the 
carbohydrates  mentioned  and  others  on  but  one,  two  or  three  of  them.  Some  reduce  nitrates 
and  others  do  not. 


FIG.   n. — SPREAD    FROM    Pus    SHOWING    STAPHYLOCOCCI.     STAINED  BY    GRAM'S    METHOD 
(4  X  eyepiece  and  Ma  oil  immersion.) 


**"   w^*   '  .* 

*     *         V 


FIG.   12. — STAPHYLOCOCCI  IN  SPREAD  FROM  A  CASE  OF  NON-SPECIFIC  URETHRITIS.     STAINED 

BY  GRAM'S  METHOD. 

(4  X  eyepiece  and  y\2  oil  immersion.) 


STAPHYLOCOCCI  57 

growth  covers  the  surface  in  several  days  and  is  white,  golden  or  lemon-colored, 
according  to  the  staphylococcus  present. 

Indol  is  not  produced  in  48  to  72  hours. 

Media  Containing  dextrose,  lactose,  saccharose,  maltose,  mannite  or  glycerin 
may  not  be  acidulated,  usually  they  are,  but  gas  is  not  formed. 

Spore  Formation. — Staphylococci  do  not  form  spores. 

Resistance. — Staphylococci  remain  alive  on  culture  media  for  months. 
Deprived  of  moisture  they  remain  for  several  weeks  or  months.  Repeated 
freezing  does  not  kill  them.  When  surrounded  by  or  contained  in  albuminous 
matter  they  are  especially  resistant.  Marked  variations  in  resistance  to 
chemical  and  thermal  germicides  are  shown  by  different  cultures;  1:1000 
solutions  of  bichloride  of  mercury  kills  them  in  10  minutes;  i:  100  carbolic  acid 
kills  in  from  J^  to  2  hours.  In  a  moist  state  they  are  usually  killed  by  an  ex- 
posure of  J/2 -hour  to  6o°C.;  some  strains  resist  8o°C.  for  i  hour.  Boiling  kills 
them  almost  instantly.  In  a  dry  state,  9o°C.  to  ioo°C.  for  J£  hour  is  required 
to  kill  them. 

Toxin. — Staphylococci  produce  an  intracellular  toxin  destructive  to  both  red 
and  white  blood  cells. 

Agglutinins. — Specific  for  the  staphylococcus  have  been  produced  experi- 
mentally, but  cannot  be  regularly  detected  in  patients  infected  with  Staphy- 
lococci. 

PATHOGENESIS 

Staphylococci  vary  in  virulence,  some  are  very  virulent  and  others  are  in- 
capable of  producing  disease.  The  majority  of  infections  following  wounds  and 
operations  and  most  of  the  suppurative  processes,  boils  and  abscesses  are  caused 
by  Staphylococci,  usually  staphylococcus  aureus.  No  portion  of  the  body  is 
invulnerable  to  them.  They  are  sometimes  the  offending  organisms  in  rhinitis, 
otitis,  coryza,  pharyngitis,  bronchitis,  pneumonia,  pleurisy,  endocarditis,  syno- 
vitis,  enteritis,  nephritis,  cystitis,  urethritis  and  meningitis.  Staphylococci 
infections  may  be  localized  or  widespread.  Entering  a  wound  they  may  lodge 
in  adjacent  tissue,  injure  or  destroy  it,  but  progress  no  further,  as  in  single 
abscess  formation.  They  may  enter  through  a  wound,  invade  the  lymph  glands, 
travel  through  the  lymph  vessels  and  lodge  in  various  tissues  and  organs  produc- 
ing inflammation  or  suppuration  at  each  focus.  Likewise,  they  may  enter  the 
blood  stream  and  be  disseminated  throughout  the  body  or  lodge  in  one  or 
several  organs. 

Lower  animals  seem  less  susceptible  to  staphylococcus  infection  than  man, 
but  they  are  not  immune. 

Cultures  injected  into  guinea-pigs,  rats,  rabbits,  cats  or  dogs  act  as  they  do 
in  man.  Of  the  animals  mentioned  rabbits  are  most  susceptible  to  Staphylococci. 

DIAGNOSIS 

Upon  inflamed  surfaces  and  in  pus  produced  by  Staphylococci  the  organisms 
are  abundant.  A  loopful  of  pus,  or  scrapings  from  an  inflamed  surface,  ob- 
tained with  a  cotton  swab,  when  smeared  on  a  slide,  dried  and  stained,  will 


58  MEDICAL  BACTERIOLOGY 

disclose  the  nature  of  a  staphylococcus  infection;  it  does  not  indicate  which 
staphylococcus  is  the  offender;  to  determine  this  cultures  on  agar  or  gelatin 
must  be  made  and  chromogenesis  observed. 

When  infection  of  the  blood  is  suspected  a  conspicuous  vein  is  sought,  usually 
at  the  elbow,  the  skin  overlying  the  vein  is  asepticized,  a  sterile  needle  is  thrust 
into  the  vein,  and  from  2  to  10  cc.  of  blood  withdrawn  with  a  sterile  glass  syringe. 
The  blood  is  immediately  ejected  into  a  flask  containing  200  cc.  of  sterile  bouillon. 
The  flask  is  shaken  and  then  incubated  at  37°C.  for  i  or  2  days.  If  growth  ap- 
pears a  loop  full  of  the  bouillon  is  smeared  on  a  slide,  stained  and  examined; 
subcultures  on  agar  and  gelatin  may  be  made. 

When  obtaining  material  from  an  inflamed  surface,  pus,  blood  or  urine  to 
examine  for  staphylococci,  the  common  presence  of  staphylococci  upon  the  sur- 
face of  the  body  must  be  remembered  and  scrupulous  precaution  observed  to 
prevent  contamination,  otherwise  misleading  findings  may  be  made. 

Opsonic  index  and  complement  fixation  tests  have  a  limited  value  in  the 
diagnosis  of  obscure  staphylococcus  infections,  but  are  seldom  employed. 

SERUM  AND  VACCINE  THERAPY 

Sera  have  been  prepared  to  combat  staphylococcus  infections,  but  their 
value  is  slight.  Vaccines  or  bacterins  fortify  or  elevate  natural  immunity  and 
so  modify,  limit  and  curtail  infections,  in  favorable  cases. 


CHAPTER  VIII 
STREPTOCOCCI 

Streptococci  occur  in  air,  water  and  soil,  occasionally  in  horse  manure  and 
cow  manure,  sometimes  on  the  skin  and  in  the  mouth  of  healthy  people. 

Morphology. — Streptococci  are  spherical,  unicellular  organisms  from  0.5 
to  i. on  in  diameter.  They  are  arranged  in  chains.  These  chains  may  be  short 
or  long,  composed  of  three  or  four  cocci,  a  dozen  or  more;  they  may  be  straight, 
curved  or  tangled,  especially  the  longer  chains.  Streptococci  found  in  blood, 
spinal  fluid,  and  cultures  from  them  often  appear  in  pairs,  but  on  further  cultiva- 
tion assume  their  regular  arrangement  in  chains. 

Streptococci  are  non-motile,  stain  readily  with  anilin  stains  and  are  Gram 
positive. 

Growth. — Streptococci  grow  well  on  ordinary  culture  media,  having  a 
slightly  acid,  neutral  or  slightly  alkaline  reaction.  The  optimum  temperature 
for  growth  is  37°C.,  but  though  slowly,  growth  takes  place  at  any  temperature 
between  i5°C.  and  44°C. 

Agar. — Surface  growth  on  agar  appears  in  1 8  to  24  hours.  The  colonies  are 
convex,  raised,  grayish-white,  opalescent  and  have  lace-like,  slightly  corrugated 
edges.  They  are  pin-point  in  size  and  usually  do  not  coalesce  nor  enlarge  as 
the  culture  ages;  after  several  days  they  tend  to  die  out. 

Gelatin. — Surface  growth  on  gelatin  is  the  same  as  that  on  agar,  with  the 
exception  that  colonies  are  more  distinctly  white. 

Stab  cultures  in  gelatin  develop  slowly,  small,  round,  opaque,  white  colonies 
appear  along  the  stab  in  from  24  to  48  hours,  and  the  culture  dies  when  5  or  6 
days  old.  With  the  exception  of  the  streptococcus  fecalis  and  possibly  a 
few  pathogenic  streptococci,  they  do  not  liquefy  gelatine. 

Blood  Serum.— On  coagulated  blood  serum  growth  is  the  same  as  on  agar, 
except  that  it  is  usually  more  luxuriant. 

Potato. — Growth  apparent  to  the  naked  eye  seldom,  if  ever,  occurs  on  this 
medium. 

Milk  is  acidulated;  some  streptococci  coagulate  it  and  others  do  not. 

Bouillon. — Cultures  incubated  at  37°C.  show  a  light,  white  sediment  in  24 
hours;  at  first  it  sticks  to  the  sides  of  the  tube,  later  it  falls  to  the  bottom,  in 
most  cases  leaving  the  medium  clear.  Lactic  acid  is  formed.  In  bouillon 
streptococci  find  most  favorable  conditions  for  development  of  long  chains. 

Both  bouillon  and  agar  containing  blood  serum  or  ascitic  fluid  favor  a  more 
luxuriant  growth  of  streptococci  than  plain  bouillon  and  agar. 

Blood  agar  (2  parts  agar  mixed  with  5  parts  whole  human  blood).  On  this 
medium  different  strains  of  streptococci  show  slight  differences  in  growth. 

59 


60  MEDICAL  BACTERIOLOGY 

Schottmiiller  has  employed  this  medium  to  differentiate  streptococci  into  several 
groups : 

Streptococcus  pyogenes  or  erysipelatos,  grayish  colonies  surrounded  by  a 
zone  of  hemolysis. 

Streptococcus  viridans,  greenish  colonies,  very  slight,  if  any,  hemolysis. 

Streptococcus  mucosus,  slimy  colonies,  no  hemolysis. 

Streptococci  do  not  form  spores  and  do  not  produce  indol. 

Powers  of  Resistance. — Streptococci  are  much  less  resistant  to  germicidal 
agents  than  staphylococci.  Pathogenic  forms  lose  their  virulence  by  cultiva- 
tion on  culture  media;  cultures  die  out  in  the  course  of  days  or  weeks;  drying 
rapidly  reduces  virulence  and  kills  in  the  course  of  weeks;  at  i2o°C.  in  hot-air 
sterilizer  they  are  destroyed  in  J^  hour;  in  a  moist  state  exposure  to  6o°C.,for 
from  }4  to  i  hour  kills;  boiling  is  almost  instantly  destructive.  Saprophytic 
forms  are  more  resistant. 

TOXIN 

Streptococci  possess  a  variable  intracellular  toxin;  attempts  to  produce  an 
antitoxin  have  been  futile. 

Agglutinins  do  not  appear  in  the  blood  of  those  infected  with  streptococci. 
Experimentally,  animals  can  be  made  to  produce  agglutinins,  but  agglutination 
tests  are  unsatisfactory  in  diagnosis  and  bacteriological  classification.  The 
same  is  true  regarding  amboceptors  and  complement  fixation  tests. 

Antistreptococci  sera  have  not  given  satisfactory  therapeutic  results. 
Bacterial  vaccines  are  of  value  in  combatting  infection  and  increasing  resist- 
ance to  streptococci,  in  selected  cases. 

PATHOGENESIS 

Streptococci,  like  staphylococci,  are  strikingly  variable  in  virulence;  some 
are  non-pathogenic,  others  but  feebly  pathogenic  and  some  are  extremely  viru- 
lent. Virulence  not  only  varies  in  different  strains  of  streptococci,  but  individ- 
uals show  marked  variations  in  pathogenicity  at  different  times.  Virulence 
is  increased  by  passage  through  animals,  especially  rabbits  and  man.  It  is 
decreased  by  cultivation  on  artificial  media  and  saprophytic  existence.  Organ- 
isms isolated  from  a  person  whom  they  killed  lose  their  pathogenic  power  to 
a  large  degree  by  cultivation  on  culture  media  for  a  long  time.  If  such  an  at- 
tenuated culture  is  passed  through  several  rabbits  its  original  virulence  may  be 
restored. 

Streptococci  may  be  associated  with  disease  either  as  its  exciting  cause  or  as 
secondary  invaders,  complicating  or  aggravating  a  condition  that  started  as  a 
staphylococcus  infection,  tuberculosis  or  other  infection. 

Streptococcus  infections,  like  staphylococcus  infections,  may  be  localized, 
the  organisms  confined  to  a  circumscribed  portion  of  the  body,  or  they  may  be 
septicemic,  the  organisms  pervading  the  blood-stream. 

Any  portion  or  tissue  may  be  involved;  as  a  rule,  localized  streptococcic 
lesions  show  a  greater  area  of  inflammation  and  edema  in  proportion  to  the 


FIG.  13. — STREPTOCOCCUS  IN  A  SMEAR  FROM  Pus.     STAINED  WITH  METHYLENE  BLUE. 

(4  X  eyepiece  and  y\i  oil  immersion  objective.) 


STREPTOCOCCI  6 I 

amount  of  suppuration  that  occurs  than  do  staphylococcic  lesions,  and  the  pus 
is  more  serous  in  character. 

Some  cases  of  broncho-penumonia,  meningitis  and  angina  are  caused  by 
streptococci.  Streptococci  are  widely  believed  to  be  the  specific  cause  of 
erysipelas ;  they  are  the  common  cause  of  puerperal  septicemia,  and  though  it  is 
very  doubtful,  some  believe  they  are  the  specific  cause  of  scarlet  fever,  in  which 
disease  they  are  usually  found  present  in  the  mouth  and  throat.* 

Though  not  generally  accepted  at  this  time,  one  must  bear  in  mind  the  teach- 
ings of  Rosenow  that  streptococci  may  mutate  and  become  pneumococci  and 
vice  -versa.  Rosenow  strongly  believes  that  the  location  of  streptococci  when 
they  have  entered  the  human  body  and  the  character  of  the  disease  which  fol- 
lows is  determined  by  the  particular  strain  of  streptococcus  infecting;  different 
strains  manifesting  more  or  less  constant  selectivity  or  predilection  to  attack 
special  organs  or  tissues,  e.g.,  one  strain  will  affect  the  appendix,  another  the 
heart,  another  the  stomach,  etc. 

Rosenow  and  others  believe  epidemic  poliomyelitis  is  caused  by  streptococci. 

DIAGNOSIS 

Erysipelas  and  other  inflammatory  conditions,  where  pus  is  scant  or  absent, 
usually  show  some  edema;  by  making  a  small  incision  in  an  appropriate  place 
serous  fluid  may  be  obtained  with  a  sterile  capillary  tube.  From  this  several 
smears  are  made.  These  are  stained  and  examined  and  will  usually  be  found 
sufficient  to  establish  the  diagnosis  in  doubtful  conditions  caused  by  strepto- 
cocci. However,  organisms  may  not  be  found  in  the  smears,  or  the  morphology 
may  be  such  as  to  make  it  impossible  to  say  whether  streptococci  or  pneumococci 
were  observed;  then  it  is  desirable  to  make  cultures.  The  fluid  is  obtained  from 
the  lesion  with  a  sterile  capillary  tube  and  planted  on  several  tubes  of  media — 
agar,  blood  agar,  Loeffler's  blood  serum  or  other  media.  These  tubes  are  in- 
cubated at  37°C.  for  24  to  48  hours,  and  the  growth  examined  both  macroscopic- 
ally  and  microscopically.  Pus  and  sputum  are  examined  for  streptococci  in 
the  same  way. 

Where  streptococcus  bacteremia  exists  or  is  suspected,  blood  cultures  must 
be  made.  The  method  of  obtaining  blood  and  inoculating  media  is  the  same  as 
when  making  blood  cultures  for  staphylococci,  previously  described. 

Streptococci  arranged  in  pairs  rather  than  chains  and  sometimes  encapsu- 
lated, so  that  they  are  indistinguishable  from  pneumococci  under  the  micro- 
scope, are  occasionally  found,  most  often  in  examination  of  blood  and  sputum. 
Differentiation  requires  culture. 

Inulin  bouillon  is  fermented  by  pneumococci,  not  by  streptococci;  pneumo- 
cocci form  dry,  blackish  colonies  on  blood-agar  without  hemolysis;  streptococci 
form  whitish  or  greenish  colonies  with  or  without  hemolysis;  pneumococci  grow 
in  the  sterile  bouillon  filtrate  from  streptococcus  cultures,  streptococci  do  not. 

*  See  "Further  Experiments  with  a  Streptococcus  Isolated  from  Cases  of  Acute  Rheuma- 
tism," Beattie,  J.  M.,  Journal  of  Pathology  and  Bacteriology,  April,  1910. 

"The  Etiology  of  Erysipelas  and  Allied  Infections,"  Panton  and  Adams,  The  Lancet, 
Oct.  9,  1909. 


62  MEDICAL  BACTERIOLOGY 

In  doubtful  cases  differentiation  of  streptococci  and  pneumococci  is  difficult 
because  intermediate  forms  not  only  vary  from  classic  streptococci  and  pneu- 
mococci in  morphology,  but  also  in  cultural  characteristics,  effects  on  rabbits, 
and  behavior  when  mixed  with  agglutinins. 

SERUM  AND  VACCINE  THERAPY 

A  small  percentage  of  cases  of  streptococci  infection  is  greatly  benefited  by 
serum  treatment,  the  condition  being  improved  to  a  degree  unattainable  by 
other  measures,  but  the  majority  is  not  influenced,  and  taken  as  a  whole,  serum 
treatment  of  streptococcus  infections  is  still  unsatisfactory. 

The  same  is  true  of  vaccine  treatment  of  acute  streptococcus  infections,  but 
in  the  treatment  of  chronic  or  recurrent  streptococcus  infections  and  the  sequelae 
of  erysipelas,  autogenous  vaccines  have  a  distinct  value,  and  it  is  always  ad- 
visable to  employ  them. 

The  extensive  studies  of  streptococci  and  pneumococci  executed  in  recent 
years  by  Rosenow  and  published  in  the  Journal  of  the  American  Medical  Asso- 
ciation and  other  American  journals  of  medicine,  should  be  carefully  read  by 
advanced  students.  Among  other  things  clearly  disclosed  is  the  fact  that  in 
some  (possibly  many)  species  of  bacteria  there  are  strains  that  will  not  grow  in 
culture  media  under  ordinary  aerobic  or  anaerobic  conditions;  organisms  that 
only  grow  in  a  certian  oxygen  concentration,  the  limits  of  which  are  narrow. 

To  insure  growth  of  such  organisms  extra  long  test-tubes  are  filled  with  solid 
media  nearly  to  the  top.  Either  shake  or  stab  cultures  are  made  so  as  to  dis- 
tribute the  plant  from  top  to  bottom  of  the  tube.  Under  such  conditions  growth 
generally  occurs  in  a  zone  %  to  i  inch  in  depth,  the  medium  above  and  below 
appearing  sterile.  Such  zones  of  growth  may,  with  different  organisms,  develop 
at  any  level  from  near  the  bottom  to  near  the  top  of  the  tube. 

The  proper  oxygen  supply  or  tension  may  be  provided  for  such  sensitive 
strains  so  as  to  induce  growth  by  connecting  the  tube  planted  with  streptococci 
to  a  tube  planted  with  bacillus  subtilis,  with  a  small  hose,  in  such  a  way  that 
the  cultures  do  not  mix. 


CHAPTER  IX 

PNEUMOCOCCUS 

(Diplococcus  lanceolatus) 

.The  pneumococcus  occurs  in  the  nose  and  mouth  of  many  healthy  people, 
and  in  sputum  from  those  who  harbor  the  organism. 

Morphology . — Pneumococci  occur  chiefly  in  pairs;  they  are  not,  as  a  rule, 
spherical,  but  lancet-shaped,  one  end  oval  the  other  slightly  pointed;  they  are 
arranged  with  the  long  axis  of  each  pair  of  cocci  on  the  same  pole,  the  oval 
ends  in  apposition,  the  pointed  ends  at  the  extremities  of  the  long  axis  of  each 
pair.  Each  pair  of  pneumococci  is  surrounded  by  a  capsule  when  they  are 


FIG.  14. — PNEUMOCOCCUS,  FROM  CULTURE.     SHOWING  IN  PAIRS  AND  ALSO   A  FEW  CHAINS; 
CAPSULESjUNSTAiNED.      (4  X  eyepiece  and  y\-i  oil  immersion  objective.) 

found  in  the  body  fluids;  cultivated  upon  culture  media,  the  capsule  is  nearly 
always  lost.  This  capsule  which  surrounds  each  pair  of  cocci  appears  as  a  light 
halo  surrounding  the  organisms  stained  in  the  ordinary  way  (with  Loeffler's 
methylene  blue,  eosin,  fuchsin  or  by  Gram's);  by  special  methods  of  staining 
the  capsule  may  be  tinted. 

Pneumococci  may  be  found  in  chains,  but  their  nature  is  usually  discernible 
because  there  is  usually  a  greater  distance  between  each  pair  than  between  the 
elements  of  pairs,  the  diplococcic  nature  remaining  apparent.  Occasionally 
pneumococci  are  oval  or  almost  spherical.  Different  strains  vary  in  size,  the 
average  being  slightly  larger  than  staphylococci  or  streptococci.  The  pneumo- 

63 


64  MEDICAL  BACTERIOLOGY 

coccus  stains  with  all  the  usual  anilin  dyes,  capsule  remaining  unstained.  It  is 
Gram  positive.  There  are  various  special  methods  of  staining  to  tint  both  the 
cocci  and  surrounding  capsule. 

Growths — The  growth  of  pneumococci  on  culture  media  differs  but  slightly 
from  streptococci;  the  range  of  temperature  at  which  growth  occurs  is  more 
restricted,  from  25°C.  to  4i°C.;  on  solid  media  the  colonies  are  more  moist  and 
transparent  than  streptococci  and,  as  a  rule,  lack  the  elevated  convex  appear- 
ance of  streptococci;  under  magnification  intertwining  chains  and  the  lace- 
like  edge  of  streptococci  colonies  are  not  observed.  On  blood-agar,  colonies 
are  dry,  blackish,  and  do  not  cause  hemolysis. 

Litmus  milk  is  more  intensely  acidified  and  more  regularly  coagulated  by 
pneumococci.  Pneumococci  ferment  inulin  and  acidify  bouillon  to  a  greater 
extent  than  streptococci.  Otherwise  the  development  on  culture  media  is 
practically  indistinguishable  from  streptococci. 

Pneumococci  do  not  form  spores. 

Resistance. — Surrounded  by  sputum  or  other  viscid  fluids  pneumococci  are 
more  resistant  to  germicides  than  when  brought  into  direct  contact  with  the 
germicide;  for  this  reason  20  to  50  per  cent,  alcohol  is  more  destructive,  because 
more  penetrating  than  stronger,  less  diffusible  substances,  and  will  kill  pneumo- 
cocci in  sputum  in  less  than  an  hour.  Pneumococci  in  masses  of  sputum  dry 
out  slowly  and  remain  viable  for  several  weeks;  in  droplets,  especially  if  exposed 
to  sunlight,  they  die  in  from  a  few  hours  to  several  days.  They  withstand  low 
temperatures  for  a  long  time,  freezing  for  weeks  or  months  being  required  to 
kill  them.  Pneumococci  are  quickly  destroyed  by  chemical  germicides,  in  5 
per  cent,  carbine  or  o.i  per  cent,  bichloride  of  mercury  solutions  they  are  killed 
in  several  minutes.  Ethylhydrocuperin,  i  :  500,000  solution  inhibits  growth, 
and  1:200,000  kills  pneumococci  in  vitro  in  3  hours  at  75°C.  Morgenroth 
and  H.  F.  Moore  have  shown  that  sterilization  of  animals  infected  with  the 
pneumococcus  can  be  accomplished  with  this  agent  but  the  toxic  dose  is  so  close 
to  the  sterilizing  dose  that  the  danger  of  employing  it  precludes  its  therapeutic 
use.  In  a  hot-air  sterilizer  pneumococci  are  killed  in  less  than  %  hour  at 
i2o°C.  In  a  moist  state  a  temperature  of  from  55°C.  to  6o°C.  kills  in  less  than 
J^  hour. 

Toxin. — Pneumococci  produce  an  intracellular  toxin;  there  is  an  antitoxin 
prepared  that  can  neutralize  it. 

Agglutinins. — Can  be  produced  experimentally  but  are  not  present  in  the 
serum  of  infected  individuals  in  sufficient  quantity  to  permit  agglutination  tests 
with  the  patient's  serum  for  diagnosis. 

Antipneumococci  sera  for  therapeutic  use  were  more  unsatisfactory  prior 
to  the  work  of  Cole  than  at  present. 

Complement  fixation  tests  are  not  employed  in  diagnosis. 

Bacterial  vaccines  do  not  confer  immunity  but  are  valuable  adjuncts  in  the 
treatment  of  certain  cases. 

Pathogenesis. — Pneumococci  have  their  virulence  increased  by  passage 
through  animals  and  lessened  by  cultivation  or  existence  outside  the  animal 


PNEUMOCOCCUS  65 

body.  It  is  believed  that  pneumococci  which  have  been  present  in  the  mouth, 
nose  or  throat  for  aconsiderable  time  without  producing  any  ill  effect,  cah  sud- 
denly become  virulent  and  cause  pneumonia.  Pneumonia  caused  by  this 
organism  ordinarily  is  not  contagious,  but  at  times  there  occur  small  epidemics 
that  seem  to  indicate  a  high  degree  of  contagiousness.  Some  epidemics  of 
pneumonia  caused  by  the  pneumococcus  are  strikingly  more  fatal  than  others. 
None  of  these  phenomena  have  been  explained. 

It  is  claimed  by  some  that  the  pneumococcus  is  the  offending  organism  in 
90  per  cent,  of  all  cases  of  acute  lobar  pneumonia  and  in  from  50  to  80  per  cent, 
of  all  other  forms  of  pneumonia.  It  is  an  established  fact  that  the  pneumococcus 
is  the  most  common  cause  of  pneumonia,  but  the  figures  given  are  probably 
too  high. 

In  pneumonia  the  organism  is  present  in  the  sputum  and  can  frequently  be 
obtained  from  the  peripheral  blood. 

The  pneumococcus  is  frequently  the  cause  of  otitis  media,  sometimes  the 
cause  of  rhinitis  and  ozena,  pleurisy,  peritonitis,  endocarditis,  pericarditis  and 
meningitis.  If  present  in  the  lachrymal  secretion  at  a  time  when  trauma  or 
operation  causes  a  breach  in  the  cornea  or  sclera,  a  serious  infection  often 
ensues. 

When  injected  subcutaneously  into  rabbits  a  localized  area  of  inflammation 
with  much  edema  or  suppuration  at  the  point  of  inoculation  may  be  the  only 
effect,  more  commonly  a  fatal  septicemia  and  death  within  48  hours  is  the 
result.  Under  certain  conditions,  lobar  pneumonia  has  been  produced  by  spray- 
ing pneumococci  into  the  lungs  of  rabbits  and  dogs. 

Diagnosis. — In  pneumonia  microscopic  examination  of  the  sputum  fre- 
quently is  the  only  procedure  required  to  establish  the  bacteriological  diagnosis; 
blood  culture  is  sometimes  required  and  is  carried  out  in  detail  as  described  for 
streptococci. 

In  localized  lesions,  such  as  pleurisy,  the  organism  is  found  in  the  exudate. 
In  meningitis  the  pneumococci  are  found  in  the  spinal  fluid. 

In  their  efforts  to  produce  a  therapeutic  serum,  Dochez,  Avery,  Cole  and 
others  recently  disclosed  numerous  important  facts  relative  to  pneumococci  and 
pneumonia.  They  have  shown  that  organisms  identical  in  morphology,  stain- 
ing and  cultural  characteristics  may  be  divided,  by  agglutination  tests,  into  four 
groups  or  types  that  differ  in  distribution,  pathogenicity  and  susceptibility 
to  immune  serum.  Experiments  have  shown  that  among  the  various  animals 
treated  with  these  organisms  horses  produce  the  most  effective  therapeutic  sera, 
and  that  the  active  principle  of  such  sera  is  confined  to  the  globulin  portion. 

Animals  inoculated  with  Type  I  pneumococci  yield  serum  which  agglutinates 
Type  I  organisms  but  does  not  agglutinate  any  others;  those  inoculated  with 
Type  II  pneumococci  yield  serum  that  agglutinates  Type  II  organisms  but  does 
not  agglutinate  any  others,  animals  inoculated  with  Type  III  pneumococci 
yield  serum  which  cannot  agglutinate  Type  III  organisms  until  they  have  been 
divested  of  their  capsule;  and  does  not  agglutinate  any  other  organisms. 

Type  IV  really  is  not  a  type — it  is  a  heterogeneous  group  to  which  is  rele- 


66  MEDICAL  BACTERIOLOGY 

gated  all  pneumococci  that  are  not  agglutinated  by  Type  I  or  Type  II  serums  and 
lack  the  capsule,  or  fail,  when  denuded,  to  agglutinate  with  Type  III  serum. 

Any  Type  IV  pneumococcus  when  injected  into  an  animal  will  produce  a 
serum  that  will  agglutinate  cultures  of  the  particular  organism  with  which 
the  animal  was  injected  but  will  not  agglutinate  other  Type  IV  pneumococci 
nor  any  other  organisms. 

The  evidence  so  far  accumulated  shows  that  Type  I  serum  administered  to 
patients  infected  with  Type  I  pneumococci  and  Type  II  serum  administered  to 
patients  infected  with  Type  II  pneumococci  exerts  a  more  beneficial  effect  on  the 
course  and  termination  of  the  disease  than  any  other  therapeutic  agent.  An 
effective  serum  for  the  treatment  of  patients  infected  with  Type  III  pneumococci 
has  not  been  produced;  the  same  is  true  of  patients  infected  with  Type  IV 
organisms. 

Type  I  serum  has  no  effect  on  disease  caused  by  any  other  organisms ;  Type 
II  serum  has  no  effect  on  disease  caused  by  any  other  organisms;  a  polyvalent 
serum  that  might  be  employed  with  some  benefit  in  any  case,  regardless  of  the 
type  of  pneumococcus  offending,  while  most  desirable,  has  not  yet  been  produced. 

Such  observations  as  have  been  made  during  the  last  2  years  seem  to  show 
that  the  majority  of  cases  of  acute  lobar  pneumonia  are  caused  by  Type  I  or 
Type  II  pneumococci;  that  in  different  localities  and  years  there  are  variations 
in  the  predominance  of  these,  at  one  time  Type  I  infections  are  more  numerous 
than  Type  II  and  vice  versa;  that  Type  IV  infections  constitute  about  25  per  cent, 
of  all  cases;  that  Type  III  is  the  most  virulent,  Type  II  next  in  virulence  and 
Type  IV  least;  that  the  pneumococci  found  in  the  mouth  or  sputum  of  healthy 
and  tuberculous  persons,  in  practically  all  cases  are  Type  IV  organisms;  and  that 
the  more  virulent  forms  are  present  in  the  sputum  of  convalescent  patients  for 
a  comparatively  short  time,  as  a  rule. 

It  now  seems  desirable  to  discover  to  which  of  the  four  types  the  offending 
organism  belongs  in  every  pneumococcus  infection  and  for  this  purpose  a  com- 
paratively simple  technique  has  been  worked^out. 

IDENTIFICATION  OF  DIFFERENT  TYPES  OF  PNEUMOCOCCI 

Wash  out  patient's  mouth  with  sterile  water;  have  patient  cough  up  some  ' 
mucus  from  the  throat  or  lungs;  catch  this  sputum  in  a  sterile  container,  put 
about  7  cc.  of  it  in  a  centrifuge  tube,  add  an  equal  quantity  of  sterile  water, 
shake,  centrifugalize  at  high  speed  for  15  minutes.  Remove  supernatant  fluid, 
fill  tube  up  again  with  sterile  water,  shake,  centrifugalize  at  high  speed  for  15 
minutes,  discard  supernatant  fluid. 

Draw  the  sediment  into  a  sterile  syringe  and  inject  ^  cc.  into  the  peritoneal 
cavity  of  a  mouse;  from  3  to  6  hours  later  chloroform  the  mouse,  inject  10  cc. 
of  normal  salt  solution  into  its  peritoneal  cavity  (to  collect  the  organisms  that 
have  multiplied)  and  withdraw  it  again.  This  fluid  is  centrifugalized  just 
enough  to  free  it  of  tissue  cells. 

Place  five  test-tubes  in  a  rack  and  put  an  equal  quantity  of  the  washings 
from  the  mouse  in  each  of  the  first  three  tubes — none  in  the  last  two.  Put 


PNEUMOCOCCUS  67 

Type  I  pneumococcus  serum  in  the  first  and  fourth  tubes.  Put  Type  II  pneumo- 
coccus  serum  in  the  second  and  last  tubes. 

Serum  is  added  in  the  proportion  of  i  part  serum  to  29  parts  of  culture. 

Add  salt  solution  sufficient  to  make  the  quantity  of  fluid  in  all  the  tubes 
the  same.  Shake  each  tube  to  mix  its  contents  and  incubate  at  37°C.  for  2 
•  hours  or  longer. 

If  agglutination  occurs  a  whitish  clump,  like  sputum,  forms  in  the  bottom 
of  the  tube,  the  supernatant  fluid  being  clear.  If  agglutination  does  not  occur 
there  is  no  change  in  the  appearance. 

When,  for  any  reason,  it  is  impossible  or  undesirable  to  obtain  the  offending 
organism  from  the  patient's  sputum,  a  sterile  glass  syringe,  fitted  with  a  suitable 
needle  is  used  to  withdraw  fluid  from  the  affected  lung;  fluid  so  obtained  is 
immediately  transferred  to  a  tube  or  flask  containing  bouillon  and  this  is  in- 
cubated at  37°C.  for  12  to  18  hours;  as  growth  appears  the  broth  culture  is. 
employed  the  same  as  the  washings  from  a  mouse,  in  making  agglutination  test. 
Blood  cultures,  when  procured,  may  be  used  likewise. 

At  the  present  time  the  identification  of  the  form  of  pneumococcus  producing 
pneumonia  is  being  made  by  mixing  equal  quantities  of  patient's  urine  and 
serum.  A  positive  reaction  is  indicated  by  turbidity  within  a  few  minutes 
after  mixture.  This  test  should  be  made  within  a  few  minutes  after  the  urine 
has  been  withdrawn  and  after  preliminary  centrifugalization  of  the  urine  to 
make  it  perfectly  clear. 

SERUM  THERAPY 

Cole  believes  serum  treatment  should  not  be  employed  until  agglutination 
tests  have  been  made;  that  Type  I  serum  should  only  be  given  to  patients  from 
whom  Type  I  organisms  have  been  isolated  and  that  Type  II  serum  should  only 
be  given  to  patients  from  whom  Type  II  organisms  have  been  isolated,  that  serum 
treatment  should  not,  at  present,  be  attempted  when  Type  III  or  Type  IV 
pneumococci  are  the  offenders. 

When  serum  treatment  is  indicated,  the  earlier  in  the  disease  it  is  employed 
the  more  beneficial  its  effect. 

"When  admitted,  the  patient  was  given  0.5  cc.  of  serum  subcutaneously  to 
discover  if  hypersensitiveness  existed.  As  soon  as  the  type  of  organism  was 
determined,  from  50  to  100  cc.  of  the  serum,  diluted  one-half  with  salt  solution, 
were  injected  intravenously. 

"The  condition  of  the  patient  served  as  a  guide  in  the  later  treatment. 
Usually  the  serum  was  not  administered  oftener  than  every  12  hours.  The 
patients  treated  received  totals  of  from  190  to  460  cc.  of  serum,  except  one,  who 
received  a  total  of  700  cc.  of  serum." 


CHAPTER  X 
MENINGOCOCCUS 

(DIPLOCOCCUS  INTRACELLULARIS  MENINGITIS, 
WEICHSELBAUM) 

The  meningococcus  occurs  in  the  nose  and  perhaps  the  throat  of  some  ap- 
parently healthy  persons  and  occasionally  in  the  nose  of  one  suffering  with 
meningitis. 

Morphology. — The  meningococcus  is  about  the  same  size  as  staphylococci, 
but  marked  variations  in  size  are  observed  in  organisms  obtained  from  the  same 
source.  They  are  arranged  in  pairs  and  are  shaped  like  a  coffee  bean,  oval  with 
flat  sides  in  apposition.  The  meningococcus  stains  with  all  the  usual  anilin 
dyes  and  is  Gram  negative.  A  peculiarity  in  staining  sometimes  observed  is 
that  different  organisms  in  the  same  smear  vary  in  staining,  some  being  faintly 
stained  and  others  deeply  stained.  Some  observers  have  noted  granular  stain- 
ing, one  portion  of  a  coccus  staining  more  deeply  than  another. 

The  meningococcus  is  non-motile. 

Growth. — The  meningococcus  is  an  obligate  aerobic  organism.  Develop- 
ment on  culture  media  occurs  only  at  temperatures  between  25°C.  and  42°C.; 
best  at  37°C.  When  a  number  of  tubes  of  culture  media  are  planted  with  men- 
ingococci  obtained  from  the  nares  or  spinal  fluid  only  a  few  show  growth. 
Meningococci  which  have  led  a  saprophytic  existence  on  culture  media  and 
have  been  transplanted  several  times  possess  a  greater  aptitude  to  grow  on 
culture  media;  cultures  from  them  nearly  always  show  growth.  Several  cubic 
centimeters  of  pus  or  spinal  fluid  containing  meningococci  must  be  planted  on 
media  to  obtain  growth,  even  when  microscopic  examination  discloses  the  pres- 
ence of  many  organisms  in  the  fluid. 

Wherry  and  Oliver,  also  Cohen  and  Markle,  state  that  partial  oxygen  ten- 
sion cultures,  made  by  connecting  culture  tubes  with  slant  agar  cultures  of 
bacillus  subtilis,  with  rubber  hose,  show  abundant  growth  of  meningococci  and 
gonococci  when  aerobic  cultures  show  scant  and  anaerobic  cultures  no  growth. 

Agar. — If  growth  occurs,  transparent,  pin-point-sized,  round  colonies  appear 
in  24  hours,  they  become  grayish  and  opaque  in  the  center  and  remain  discrete. 
After  several  days  they  die. 

Glycerin  agar  shows  the  same  character  of  growth  as  plain  agar. 

Blood-serum  Agar,  ascitic-fluid  agar  and  blood-smeared  agar  are  more 
favorable  media  for  cultivation  than  plain  agar  and  show  a  more  luxuriant 
growth. 

Loeffler's  blood  serum  is  the  best  solid  medium  for  cultivation  of  the 
meningococcus.  Pin-point  grayish  colonies  appear  in  24  hours,  as  on  agar;  the 
growth  may  become  confluent. 


MENINGOCOCCUS  69 

Plain  bouillon  is  not  a  favorable  medium  upon  which  to  cultivate  the 
meningococcus;  when  growth  does  occur  it  is  scant  and  similar  to  that  observed 
in  bouillon  which  contains  serum  or  ascitic  fluid. 

Serum  Bouillon. — Slight  cloudiness  in  24  hours;  after  36  to  48  hours  a  scant 
grayish-pellicle  forms. 

Milk  is  neither  acidulated  nor  coagulated. 

All  cultures  of  the  meningococcus  die  out  in  from  3  to  6  days,  as  a  rule,  but 
those  which  have  been  cultivated  for  a  long  time  may  survive  longer  than  a  week 
without  transplanting. 

Glucose  and  maltose  are  the  only  sugars  fermented  by  this  organism. 

Resistance. — Drying  kills  the  meningococcus  in  several  hours,  low  tempera- 
tures and  freezing  kill  in  several  days,  i :  1000  bichloride  and  i  per  cent,  phenol 
solutions  kill  in  less  than  2  minutes.  In  the  hot-air  sterilizer  ioo°C.  for  15 
minutes  and  in  the  moist  state  6o°C.  for  10  to  15  minutes  kills  meningococci. 
Their  resistance  outside  the  body  is  slight. 

Toxin. — The  meningococcus  produces  an  intracellular  toxin  of  much 
virulence. 

Agglutinins  appear  in  the  blood  of  persons  having  meningococcus  meningitis 
and  can  be  produced  by  injecting  attenuated  or  killed  cultures  into  animals. 
Complement  fixation  tests  are  employed  to  differentiate  between  meningococcus, 
gonococcus  and  micrococcus  catarrhalis,  but  seldom  in  diagnosis. 

Antimeningococcus  serum  is  the  most  valuable  agent  employed  in  the  treat- 
ment of  meningococcus  infections. 

Pathogenesis. — In  man  the  meningococcus  causes  meningitis,  inflammation 
of  the  naso-pharynx  and  throat.  Some  observers  have  reported  finding  the 
organism  in  the  blood  of  persons  afflicted  with  meningitis.  Conditions  similar 
to  those  occurring  in  man  can  be  produced  by  inoculation  into  monkeys.  The 
organism  is  pathogenic  for  white  mice,  injected  into  the  peritoneal  cavity  it 
produces  a  fatal  peritonitis  with  exudate  that  contains  many  organisms. 
Other  animals  are  immune  or  suffer  from  toxemia  when  inoculated. 

Recent  investigations  have  shown  there  are  numerous  strains  of  meningo- 
cocci and  para-meningococci  which  fail  to  agglutinate  with  serum  from  animals 
inoculated  with  the  strains  most  commonly  isolated  from  patients  with  menin- 
gitis— strains  that  are  not  acted  upon  by  therapeutic  sera  on  the  market,  hence 
the  desirability  of  polyvalent  sera. 

Diagnosis. — In  carriers  the  meningococcus  is  present  on  the  mucosa  of  the 
naso-pharynx  or  throat.  Suspected  carriers  are  examined  by  passing  a  sterile 
cotton  swab  over  these  surfaces  and  then  planting  several  tubes  of  blood  serum 
and  agar  with  the  swab.  Smears  for  microscopic  examination  are  made  from 
the  swab. 

When  the  disease  is  epidemic  cases  of  naso-pharyngitis  are  examined  as 
carriers,  for  it  is  believed  that  the  meningococcus  first  assaults  these  membranes. 
Its  activity  may  be  confined  to  them  or  it  may  pass  to  the  meninges  and  produce 
meningitis. 

In  cases  of  meningitis  caused  by  the  meningococcus  the  organism  in  nearly  all 


7O  MEDICAL  BACTERIOLOGY 

cases  may  be  found  in  the  spinal  fluid  and  exudate  upon  the  membranes.  The 
organisms  are  nearly  all  found  within  the  pus  cells,  polynuclear  leucocytes, 
some  of  these  cells  are  full  of  cocci.  These  coffee  bean-shaped,  Gram  negative, 
diplococci  vary  in  size.  Variations  in  size  may  be  noted  among  those  observed 
in  a  single  pus  cell;  on  an  average  they  are  somewhat  larger  than  gonococci. 

When  spinal  fluid  is  to  be  examined  a  strong  needle  about  6  inches  long, 
that  will  not  break  if  bent,  is  thrust  into  the  spinal  canal  between  the  third  and 
fourth  lumbar  vertebrae,  about  %  inch  to  one  side  of  the  median  line,  while  the 
patient  is  lying  on  his  side  with  thighs  and  head  strongly  flexed. 

When  the  fluid  begins  to  drop  or  flow  from  the  needle,  it  is  collected  in  a 
test-tube  that  will  carry  from  5  to  15  cc. 

Strict  aseptic  precautions  must  be  observed  throughout,  and  after  the 
needle  has  been  withdrawn,  the  wound  is  painted  with  iodine  and  covered  with 
a  sterile  dressing,  the  patient  remaining  reclined  and  at  rest,  for  at  least  8 
hours. 

SERUM  THERAPY 

A  bacteriolytic  serum  is  employed  in  the  treatment  of  this  disease,  the  usual 
method  of  administration  being  into  the  spinal  canal. 


CHAPTER  XI 
GONOCOCCUS 

The  gonococcus  occurs  in  the  genito-urinary  tract  of  carriers  and  in  the 
affected  organs  of  infected  persons.  It  is  not  found  outside  the  human  body. 

Morphology. — The  gonococcus  occurs  in  pairs,  the  cocci  arranged  like  coffee 
beans,  ovoid  with  flat  sides  in  apposition.  The  long  diameter  of  the  gonococcus 
is  from  0.6  to  0.8  p.  It  stains  with  all  the  anilin  dyes  and  is  Gram  negative. 
The  gonococcus  does  not  form  spores  and  is  non-motile.  Morphologically  and 
by  staining  this  organism  is  indistinguishable  from  the  meningococcus. 

Growth. — This  organism  cannot  be  satisfactorily  cultivated  on  plain  agar, 
gelatin  or  bouillon.  Occasionally  scant  growth  may  be  obtained  in  gelatin 
stabs  (without  liquefaction),  on  LoefHer's  serum  and  on  blood-smeared  agar; 
but  Wertheim's  medium  or  some  of  its  modifications  serve  best  for  the 
cultivation  of  the  gonococcus. 

To  tubes  containing  6  cc.  of  neutral  glycerin  agar,  liquefied  and  cooled  to 
5o°C.,  4  cc.  of  sterile  blood  serum  or  ascitic  fluid  should  be  added.  After  mix- 
ing the  serum  and  agar  the  tubes  are  slanted.  Smears  made  on  this  medium 
show  growth  in  1 8  to  24  hours  at  37°C.  The  colonies  are  round,  pin-head  size, 
whitish  and  tenacious.  After  from  3  days  to  a  week  they  usually  die  out  if 
not  transplanted. 

It  is  said  that  the  addition  of  a  piece  of  sterile  rabbit  testicle  to  culture 
medium  favors  the  propagation  of  gonococci.  While  it  is  usually  difficult  to 
obtain  a  growth  in  tubes  planted  with  gonococci  directly  removed  from  the 
human  body,  after  the  organisms  have  been  successfully  cultured  and  trans- 
planted several  times  on  media,  they  usually  grow  more  regularly,  more  abun- 
dantly and  less  exacting  as  to  the  composition  of  the  medium. 

Gonococci  will  not  grow  in  the  presence  of  free  moisture;  there  should  be 
no  water  of  condensation  in  tubes  used  for  their  cultivation. 

C.  C.  Worden  recommends  the  following  medium  for  obtaining  pure  cultures 
of  gonococci  from  material  containing  other  organisms  in  addition  to  the 
gonococci,  and  states  that  it  is  a  good  medium  for  the  cultivation  of  gonococci: 

Sodium  chloride i .  080  Gm. 

Potassium  chloride o .  045  Gm. 

Calcium  chloride 0.025  Gm. 

Sodium  bicarbonate .  0.020  Gm. 

Agar o .  250  Gm. 

Nutrient  broth 20  cc. 

Distilled  water 100  cc. 

Filter  through  cotton  into  test-tube  and  sterilize  once  in  autoclave. 
Sugars  are  not  fermented  and  indol  is  not  formed. 

71 


72  MEDICAL  BACTERIOLOGY 

Resistance. — The  gonococcus  is  a  delicate  organism,  in  dried  pus  on  linen 
and  other  articles  usually  it  dies  in  several  hours,  occasionally  it  survives  for 
several  weeks.  Freezing  for  a  number  of  days  eventually  kills,  but  tempera- 
tures between  o°C.  and  37.5°C.  do  not  destroy  it.  Temperatures  above  4o°C. 
are  injurious;  in  a  moist  state  exposure  to  6o°C.  for  10  or  15  minutes  kills. 
Drying  rapidly  destroys  gonococci;  in  a  hot-air  sterilizer  they  are  killed  at  ioo°C. 
in  a  few  minutes. 

Chemical  germicides  in  very  high  dilutions  are  rapidly  destructive. 

The  gonococcus  is  especially  sensitive  to  salts  of  silver. 

Toxin. — The  gonococcus  produces  an  intracellular  toxin. 

Agglutinins  are  not  found  in  the  serum  of  most  infected  patients;  they  may 
be  produced  by  animal  inoculation,  but  are  not  employed  in  diagnosis. 

Attempts  to  produce  antigonococcus  sera  of  therapeutic  value  have  been 
unsatisfactory. 

Vaccines  are  of  little  or  no  value  in  acute  infections ;  in  subacute  and  chronic 
infections  they  sometimes  give  brilliant  results;  the  chief  indication  for  their 
administration  is  gonococcus  arthritis. 

Pathogenesis. — The  gonococcus  has  a  predilection  for  the  mucous  mem- 
branes of  the  urethra.  It  is  most  commonly  found  as  the  cause  of  urethritis. 
Gonorrhea  is  the  most  frequent  of  venereal  diseases.  The  activity  of  this 
organism  is  not  limited  to  the  urethra  in  all  cases;  it  often  passes  further,  at- 
tacking the  cord,  testicles,  bladder  or  kidneys,  in  the  male,  and  the  bladder, 
vagina,  cervix,  uterus,  tubes  or  ovaries  in  the  female.  Occasionally  infection  is 
not  localized  in  the  genito-urinary  tract,  but  passes  to  the  blood  stream  and 
causes  endocarditis,  often  malignant;  the  gonococcus  may  be  deposited  in  the 
joints  and  cause  arthritis;  so-called  gonorrheal  rheumatism.  Infection  in 
practically  all  such  cases  is  the  result  of  sexual  intercourse  with  persons  harbor- 
ing the  organism. 

An  epidemic  form  of  vaginitis  caused  by  the  gonococcus,  not  due  to  sexual 
intercourse,  is  observed  from  time  to  time  among  female  children  in  the  wards  of 
hospitals.  The  mode  of  transmission  in  such  cases  is  not  known. 

The  gonococcus  is  sometimes  conveyed  to  the  eye  by  the  fingers  or  linen 
soiled  with  urethral  discharge  or  pus.  It  frequently  gets  into  the  eyes  of 
infants  during  their  passage  through  the  vagina  at  birth,  when  the  mother 
harbors  the  gonococcus.  In  the  eye  this  organism  produces  a  violent  inflamma- 
tion with  profuse  purulent  exudate;  not  infrequently  vision  is  impaired  or  totally 
destroyed. 

The  gonococcus  may  persist  in  the  urethra  or  vagina  long  after  manifesta- 
tions of  disease  have  ceased,  when  there  is  no  pus,  no  discharge  and  when  smears 
and  cultures  made  from  the  urethra  show  no  gonococci.  Under  these  condi- 
tions the  carrier,  individual  harboring  the  organism,  may  transmit  infection 
during  sexual  intercourse. 

The  gonococcus  does  not  infect  any  animal  other  than  man,  and  does  not 
exist  outside  the  human  body  except  under  experimental  conditions. 

Diagnosis. — In  acute  infections  of  the  genito-urinary  organs  and  the  eye, 


Katt\&r,n« 


FIG.  15.  —  GONOCOCCI    IN  URETHRAL    DISCHARGE.     (GRAM'S  STAIN.)     (From    Webster's 

"Diagnostic  Methods.") 


GONOCOCCUS  73 

gonococci  are  present  in  the  purulent  discharges.  Smears  made  from  such  dis- 
charges and  stained  by  Gram's  method  and  counter  stained  with  eosin  or  bis- 
marck  brown,  show  numerous  pus  cells,  many  of  which  contain  Gram  negative, 
coffee  bean-shaped,  diplococci;  some  pus  cells  contain  only  one  or  several  pairs, 
others  are  crowded  full  of  cocci;  some  cocci  occur  outside  pus  cells;  these  free 
cocci  may  show  isolated  pairs  here  and  there  or  several  pairs  in  a  clump.  Such 
findings,  in  the  majority  of  cases,  suffice  to  establish  a  diagnosis;  culture  is 
seldom  resorted  to  for  this  purpose.  If  gonorrhea  becomes  chronic  other  organ- 
isms frequently  invade  the  diseased  tissue  and  may  outnumber  the  gonococci 
to  such  an  extent  as  to  obscure  them.  When  the  disease  becomes  chronic  without 
secondary  infection,  and  in  carriers,  examination  of  discharge  or  secretion  may 
not  show  the  organisms.  Under  such  circumstances  the  consumption  of  several 
glasses  of  beer,  application  of  chemical  irritants  to  the  affected  part,  prostatic 
massage  or  an  orgasm  frequently  will  cause  the  appearance  of  gonococci  in 
the  urethral  secretions. 

Specific  amboceptors  are  present  and  detectable  to  a  sufficient  degree,  in 
cases  of  more  than  6  weeks'  duration,  to  make  complement  fixation  tests 
valuable  adjuncts  in  the  detection  of  occult  infections  and  hence  in  determining 
whether  clinical  cure  is  associated  with  bacteriological  sterilization. 


CHAPTER  XII 
MICROCOCCUS  CATARRHALIS 

The  micrococcus  catarrhalis  is  identical  in  appearance  with  the  gonococcus 
and  meningococcus  and,  like  them,  is  Gram  negative.  It  has  only  been  found 
so  far  on  the  mucous  membrane  of  the  respiratory  passages  and  in  the  sputum  of 
some  cases  of  pulmonary  tuberculosis  and  lobar  pneumonia  patients.  The 
organism  has  feeble  pathogenic  power  and  may  cause  catarrhal  inflammations. 
It  is  of  interest  chiefly  because  it  may  be  mistaken  for  the  meningococcus. 
These  organisms  are  easily  distinguished  by  culture. 

While  it  grows  best  at  37°C.,  the  micrococcus  catarrhalis  will  grow  at  any 
temperature  down  to  2o°C.;  the  meningococcus  will  not  grow  below  25°.  The 
micrococcus  catarrhalis  grows  well  on  all  the  ordinary  culture  media,  on  gelatin, 
agar  and  blood  serum  incubated  at  37°C.,  pin-head-sized,  round,  irregular- 
edged,  white  colonies  form  in  24  hours.  In  plain  bouillon  there  is  cloudiness 
with  a  fine  white  precipitate.  Milk  is  not  coagulated,  sugars  are  not  fermented. 


74 


CHAPTER  XIII 
MICROCOCCUS  TETRAGENUS  AND  SARCINA 

Micrococcus  tetragenus  is  found  occasionally  in  the  mouth  and  sputum  of 
healthy  people.  It  is  frequently  present  in  the  cavities  and  sputum  of  pul- 
monary tuberculosis  and  in  the  pus  from  abscesses  and  suppurative  lesions 
of  the  mouth  or  naso-pharynx. 

Morphology. — Micrococcus  tetragenus  is  recognized  by  its  arrangement  in 
fours  forming  a  square.  These  groups  of  four  are  usually  surrounded  by  a 
capsule,  but  a  capsule  is  not  always  visible.  Each  coccus  is  about  the  size  of 
the  staphylococcus.  Micrococcus  tetragenus  is  non-motile,  stains  with  all  the 
usual  dyes  and  is  Gram  positive. 

Growth. — All  conditions  favorable  to  growth  of  staphylococcus  on  culture 
media  suffice  for  cultivation  of  micrococcus  tetragenus. 

On  agar,  potato  and  blood  serum  a  luxuriant,  thick,  confluent,  white  growth 
appears  in  24  to  48  hours  at  37°C.  Small,  shiny  white  round  colonies  form  on 
gelatin;  it  is  not  liquefied. 

Milk  is  not  coagulated. 

When  cultured  on  artificial  media  micrococcus  tetragenus  usually  loses  its 
capsule  and  very  frequently  loses  its  characteristic  arrangement  in  fours  so  that 
the  appearance  of  stained  smears  from  cultures  is  often  indistinguishable  from 
staphylococci. 

Hay  infusion  culture  medium  is  said  to  favor  the  preservation  of  the  arrange- 
ment in  fours. 

Resistance. — High  and  low  temperatures  and  chemical  germicides  have  the 
same  effect  on  this  organism  as  on  staphylococci.  Oral  administration  of 
sodium  sulphite  will  cause  the  disappearance  of  sarcina  in  stomach  contents. 

Pathogenesis. — Micrococcus  tetragenus  is  primarily  a  saprophyte.  It 
usually  enters  diseased  areas  as  a  secondary  invader,  after  some  virulent  organ- 
ism has  injured  or  destroyed  tissue.  It  can  cause  abscess  formations  and  pus 
either  as  a  primary  or  secondary  invader,  usually  the  latter.  At  least  one  in- 
stance is  on  record  of  this  organism  having  entered  the  blood-stream  and  causing 
septicemia.  Its  pathogenic  properties  and  virulence  are  slight. 

Injected  into  white  mice  a  fatal  septicemia  may  result.  Injected  into  rabbits 
and  other  animals  it  causes  an  inflammation  with  or  without  pus  formation, 
followed  by  rapid  recovery  in  most  cases. 

Diagnosis. — The  characteristic  arrangement  of  micrococcus  tetragenus 
suffices  for  its  recognition  in  sputum,  stomach  contents,  pus  and  other  body 
tissues  or  fluids. 

75 


7 6  MEDICAL  BACTERIOLOGY 

SARCINA 

Sarcina  are  occasionally  found  in  the  mouth  and  sputum  of  healthy  persons, 
more  frequently  when  some  chronic  disease  of  the  respiratory  tract,  as  tuber- 
culosis, exists.  They  are  frequently  present  on  foodstuffs  exposed  to  air,  dust 
and  handling,  and  in  certain  foods,  as  eggs,  undergoing  putrefaction.  Some 
forms  are  commonly  present  in  air  and  soil. 

Of  the  five  varieties  sarcina  may  be  divided  into  according  to  the  color  of 
their  growth  on  culture  media: 

Sarcina  pulmonum  (colorless). 

Sarcina  alba,  and  cervina  (white). 

Sarcina  lutea,  flava,  and  lactis  (yellow). 

Sarcina  erythromyxa,  (red). 

Sarcina  aurantiaca  (orange). 
None  are  pathogenic  for  man. 

Morphology. — Sarcina  are  non-motile  cocci  about  the  size  of  staphylococci. 
They  multiply  by  cell  division  in  three  directions  and  cocci  remain  united  after 
fission,  so  they  are  arranged  in  cubes  each  surface  of  which  presents  four  cocci 
or  multiples  of  four.  The  appearance  of  these  packets  is  often  suggestive  of  a 
bale  of  cotton. 

Staining. — Sarcina  stain  readily  with  all  the  common  dyes  and  are  Gram 
positive. 

Growth  appears,  under  aerobic  conditions,  at  37°C.  in  24  to  72  hours  on  all 
the  ordinary  media. 

Most  strains  do  not  coagulate  milk;  a  few  show  acid  but  not  gas  production 
in  glucose  media,  usually  sugars  are  not  acted  on. 


CHAPTER  XIV 

BACILLUS  OF  INFLUENZA,  KOCH-WEEK'S  BACILLUS,  AND 
BOKDET-GENGOU  BACILLUS 

The  bacillus  of  influenza  occurs  in  the  mouth  or  throat  of  some  healthy 
persons,  in  the  saliva  and  sputum  of  influenza  patients  and  in  the  droplets  of 
mucus  expelled  with  the  breath.  Outside  the  body,  when  dried,  it  dies  in  a 
few  hours. 

Morphology. — The  influenza  bacillus  is  very  small,  0.3  to  1.5  p. 

Though  usually  rod-shaped,  occasionally  they  resemble  pneumococci,  except 
that  no  capsule  is  possessed  by  the  influenza  bacillus. 

Arrangement  is  irregular. 

Bacillus  influenza  is  non-motile  and  Gram  negative. 

Growth. — The  bacillus  of  influenza  or  Pfeiffer's  bacillus  is  an  obligate 
aerobic  organism  that  grows  at  temperatures  between  26°C.  and  42° C.,  best  at 
37°C.  It  requires  the  presence  of  hemoglobin  for  cultivation,  does  not  grow  on 
plain  bouillon,  agar  or  gelatin,  poorly  on  blood-serum  bouillon  or  serum-hemo- 
globin bouillon.  Blood-smeared  agar  is  the  best  medium  for  its  growth.  On 
this  medium  at  37°C.,  discrete  colonies  develop  in  18  to  24  hours.  Many  of 
them  are  only  visible  when  examined  microscopically,  some  are  pin-point  size. 
They  are  round,  dewdrop-like,  do  not  coalesce  and  cultures  die  out  in  about  a 
week  unless  transplanted.  Growth  is  enhanced  by  cultivation  in  symbiosis  with 
staphylococcus  aureus  and  colonies  pin-point  to  pin-head  in  size  develop. 
Spores  are  not  formed. 

Resistance.— The  bacillus  of  influenza  is  very  perishable  outside  the  body. 
In  sputum  it  may  survive  for  several  weeks,  but  usually  it  dies  in  several  days. 
Drying  kills  it  in  a  day  or  two,  ioo°C.  in  hot-air  sterilizer  kills  it  in  less  than  J^ 
hour.  Moist  heat  at  5o°C.  kills  in  less  than  %  hour.  Chemical  germicides  in 
very  weak  solutions  rapidly  destroy  it. 

Toxin. — The  influenza  bacillus  produces  an  intracellular  toxin. 

Agglutinins  are  not  found  in  the  blood  of  infected  patients  and  experi- 
mentally, agglutinin  production  is  irregular,  not  constant,  and  never  very  strong. 

Complement  fixation  tests  are  not  employed  in  the  identification  of  this 
organism. 

Pathogenesis. — The  influenza  bacillus  is  the  exciting  cause  of  some  cases  of 
coryza  and  influenza,  occasionally  it  is  the  cause  of  pneumonia  and  meningitis. 
Bacillus  of  influenza  infections  occur  sporadically,  in  epidemics  and  pandemics. 
In  some  cases  the  organism  is  present  in  the  blood  stream. 

Guinea-pigs  are  immune  to  this  organism.  Rabbits  and  white  mice  die  of 
septicemia  following  massive  intraperitoneal  inoculations.  The  pathogenicity 

77 


78  MEDICAL   BACTERIOLOGY 

of  the  influenza  bacillus  appears  to  be  exalted  by  symbiotic  development  with 
staphylococcus  aureus,  pneumococcus  and  streptococci. 

Diagnosis. — Whether  the  infection  is  localized  in  the  upper  air  passages,  or 
other  parts  are  involved,  the  organism  can  usually  be  found  in  the  nasal  secre- 
tions and  on  the  nasal  mucosa  of  infected  persons.  It  is  present  in  the  sputum 
when  the  bacillus  of  influenza  produces  pneumonia  and  in  the  spinal  fluid  when 
the  infection  affects  the  meninges.  Smears  stained  by  Gram's  method  and  cul- 
ture on  blood-smeared  agar  usually  suffice  to  establish  the  diagnosis.  In 
doubtful  cases  inoculation  of  rabbits  and  guinea-pigs  is  of  value. 

When,  as  often  occurs,  the  staphylococcus,  pneumococcus  or  streptococcus 
is  present  in  nasal  secretion,  sputum  or  spinal  fluid,  together  with  the  influenza 
bacillus,  they  suggest  a  more  serious  condition  than  uncomplicated  influenzal 
infection. 

KOCH-WEEK'S  BACILLUS 

Koch- Week's  bacillus,  sometimes  called  the  bacillus  of  acute  contagious 
conjunctivitis,  found  on  the  conjunctiva  and  in  the  lachrymal  discharge  in  cases 
of.  acute  contagious  conjunctivitis,  is  indistinguishable  from  the  influenza 
bacillus  and  is  considered  to  be  the  same  by  some  authorities. 

BOKDET-GENGOU  BACILLUS 

Bordet-Gengou  bacillus  occurs  in  pure  culture  in  the  bronchial  mucus  and 
sputum  of  some  cases  of  whooping-cough  during  the  early  days  of  the  disease; 
later  in  the  disease  other  organisms  may  also  be  present  and  make  its  recognition 
or  isolation  more  difficult.  When  present  in  pure  culture  the  bronchial  secre- 
tion shows  very  many  Bordet-Gengou  bacilli,  swarms  of  them. 

Morphology. — It  is  a  small  bacillus,  almost  as  small  as  the  bacillus  of  in- 
fluenza; it  is  oval,  sometimes  appearing  like  a  coccus;  it  stains  deepest  at  each 
end,  and  large  numbers,  close  together,  irregularly  arranged,  are  found  in  smears 
from  sputum. 

The  Bordet-Gengou  bacillus  stains  with  the  usual  anilin  dyes  and  is  Gram 
negative. 

Growth. — The  Bordet-Gengou  bacillus  does  not  grow  on  plain  agar,  gelatin 
or  bouillon.  Cultures  direct  from  bronchial  secretion  do  not  develop  so  well  as 
subcultures,  those  which  have  been  cultivated  and  transplanted  several  times. 

On  Loeffler's  blood  serum,  i  per  cent,  glycerin-potato  agar  plus  100  per  cent, 
blood  serum,  serum  agar  and  ascitic  fluid  agar,  growth  occurs  under  aerobic 
conditions  at  37°C.  Glycerin-potato  agar  serum  medium  is  the  best  for  first 
cultures  from  the  throat;  subcultures  do  well  on  any  of  the  media  mentioned. 

Microscopic  growth  develops  on  glycerin-potato  agar  serum  in  about  48 
hours.  Subcultures  show  a  faint  whitish  growth  which  is  said  to  become  more 
abundant  after  a  day  or  two. 

Diagnosis  is  based  upon  a  microscopic  examination  of  bronchial  secretion 
or  sputum. 

Vaccine  therapy  has  been  lauded  by  some  who  have  employed  it  but  is  not 
in  general  use. 


CHAPTER  XV 
BACILLUS  OF  MORAX  AND  AXENFELD 

This  organism  causes  a  subacute  or  chronic  catarrhal  conjunctivitis.  It  is 
found  in  the  exudate  or  pus  of  infected  cases.  Bacillus  of  Morax  and  Axenfeld 
is  a  diplobacillus,  arranged  in  pairs,  end  to  end.  It  is  a  large  bacillus  2  to  3  /x 
long,  0.4  IJL  wide,  has  round  ends,  stains  with  the  usual  anilin  dyes  and  is  Gram 
negative.  It  does  not  form  spores  and  is  easily  destroyed  by  heat. 

Diagnosis  is  based  upon  microscopic  examination  of  pus  or  exudate  from 
suspected  cases. 


,  79 


CHAPTER  XVI 
PNEUMOBACILLUS  OR  BACILLUS  OF  FRIEDLANDER 

BACILLUS  MUCOSUS  CAPSULATUS 

Bacillus  Friedlander  is  widely  distributed  in  nature,  it  occurs  in  air,  water, 
soil,  dust,  milk,  feces,  saliva  and  sputum;  in  the  mouth  and  sputum  of  some 
healthy  people  and  in  tissue  attacked  by  this  organism. 

Morphology. — The  pneumobacillus  is  about  i  to  4  /A  long  and  0.5  to  i.o  n 
broad,  has  rounded  ends  and  is  surrounded  by  a  distinct  capsule.  This  capsule 
is  apparent  when  organisms  removed  from  tissue  are  observed;  after  cultivation 
on  media  the  capsule  may  disappear.  Pneumobacilli  are  arranged  singly,  in 
pairs  and  in  chains.  When  in  chains  the  capsules  often  merge  as  though  one 
capsule  enclosed  the  chain.  This  bacillus  is  not  motile.  It  stains  with  all  the 
usual  stains  and  is  Gram  negative.  Special  stains  are  required  to  tint  the 
capsule.  In  preparations  stained  by  any  of  the  ordinary  methods  the  un- 
stained capsule  appears  as  a  light,  colorless  zone  surrounding  the  pigmented 
bacillus. 

Growth. — The  pneumobacillus  grows  well  on  all  the  ordinary  media  at 
temperatures  between  i5°C.  and  40°C.  Slightly  acid,  neutral  or  faintly  alkaline 
media  will  do,  but  a  slightly  acid  reaction  is  most  favorable.  Pneumobacillus 
is  an  aerobic  and  facultative  anaerobic  organism.  Bouillon  shows  growth  in 
24  to  36  hours;  it  is  clouded,  a  whitish  pellicle  forms  on  the  surface  and  later  a 
stringy  whitish  precipitate  forms. 

Gelatin  stabs  incubated  at  room  temperature  show  a  whitish  growth  in  48 
hours.  It  follows  the  line  of  the  stab  and  gradually  extends  over  the  surface, 
giving  a  tack-shaped  growth.  A  few  small  gas  bubbles  may  appear  along 
the  stab ;  the  gelatin  is  not  liquefied. 

Surface  cultures  on  gelatin  show  round,  raised,  grayish-white  colonies  in 
2  or  3  days. 

Agar. — In  24  to  36  hours  a  confluent,  moist,  white,  tenacious  growth  covers 
the  surface.  On  serum  agar  and  LoefHer's  medium  growth  is  the  same  as  on 
agar. 

Milk  is  usually  coagulated,  rapidly  by  some  strains  and  slowly  by  others; 
occasionally  pneumobacilli  are  encountered,  which  do  not  coagulate  milk,  or 
at  least  fail  until  subcultured. 

Indol  is  not  formed.  Some  pneumobacilli  ferment  all  the  sugars;  another 
group  ferments  all  the  sugars  except  lactose,  and  a  third  group  ferments  all 
sugars  except  saccharose. 

The  pneumobacillus  does  not  form  spores. 

80 


BACILLUS    OF   FRIEDLANDER  8 1 

Resistance. — The  pneumobacillus  resists  drying  for  a  long  time;  an  exposure 
for  i  hour  in  a  hot-air  sterilizer  at  ioo°C.  kills;  in  a  moist  state  60°  to  7o°C.  for 
J^  hour  kills.  When  not  surrounded  by  sputum  or  albuminous  matter  carbolic 
acid  (i  per  cent,  to  5  per  cent,  sol.)  and  i  :  1000  bichloride  solution  kill  in  a 
few  minutes. 

Toxin. — Filtrates  of  bouillon  cultures  are  toxic. 

Agglutinins  are  not  found  in  the  blood  of  infected  patients,  but  may  be  pro- 
duced by  inoculation  of  animals  with  killed  cultures;  results  are  uncertain  and 
when  an  agglutinin  is  obtained  it  is  specific  for  the  particular  strain  of  pneumo- 
bacillus used  to  produce  it,  it  does  not  agglutinate  other  pneumobacilli,  conse- 
quently agglutination  tests  are  not  of  value  in  diagnosis.  The  same  is  true  of 
complement  fixation  tests. 

Friedlander's  bacillus  has  been  found  the  causative  organism  in  pneumonia, 
septicemia,  pleurisy,  pericarditis,  peritonitis,  meningitis,  stomatitis,  tonsillitis 
and  parotiditis.  It  can  produce  suppuration  and  abscess  formation  in  many 
parts  of  the  body  but  it  is  seldom  the  cause  of  pneumonia  and  more  rarely  the 
cause  of  septicemia,  inflammation  of  endothelial  tissues  and  suppuration.  As 
a  secondary  invader  it  may  be  found  in  the  discharge  from  chronic  suppurating 
lesions,  as  gleet  and  otitis. 

Rabbits  are  somewhat  resistant  or  immune  to  the  pneumobacillus;  guinea- 
pigs  and  mice  are  susceptible  when  inoculated  with  virulent  organisms,  abscess 
formation  at  the  point  of  inoculation,  septicemia  and  death  in  several  days 
follow. 

Different  strains  of  Friedlander's  bacillus  show  marked  variations  in  viru- 
lence; some  are  devoid  of  pathogenicity.  For  this  reason  and  on  account  of 
variations  in  their  effect  on  carbohydrates,  some  observers  hold  that  bacillus 
Friedlander  is  the  type  of  a  group  of  closely  allied  organisms,  some  of  which 
are  pathogenic  and  some  non-pathogenic.  Among  these  closely  allied  bacilli, 
held  by  some  to  be  typical  Friedlander's  and  by  others  distinct  entities,  may  be 
mentioned  Bacillus  of  Rhinoscleroma,  which  is  the  exciting  cause  of  some  cases 
of  chronic  nasal  catarrh,  Bacillus  Ozenae,  which  is  the  exciting  cause  of  fetid 
nasal  catarrh,  and  Bacillus  Lactis  Aerogenes,  found  in  air,  dust,  soil,  water  and 
especially  in  milk. 

Diagnosis. — When  the  pneumobacillus  is  the  cause  of  mucous  membrane 
inflammations  it  is  found  in  the  surface  exudate,  it  is  present  in  the  sputum  when 
the  exciting  cause  of  pneumonia,  in  the  exudate  of  pleurisy,  pericarditis  and 
peritonitis  and  in  the  pus  of  suppurating  lesions.  In  the  majority  of  cases 
diagnosis  can  be  made  by  examination  of  smears  stained  by  Gram's  method  and 
counterstained;  occasionally  culture  for  differentiation  is  necessary. 


CHAPTER  XVII 

DIPHTHERIA  BACILLUS 

(Klebs-Loeffler  Bacillus) 

The  diphtheria  bacillus  is  found  in  the  air,  and  dust  and  on  the  furniture  and 
drapings  of  rooms  inhabited  by  diphtheria  patients  and  diphtheria  carriers,  on 
the  clothing  and  utensils  of  diphtheria  patients  and  carriers  and  perhaps  on 
dogs,  cats  and  cows  associated  with  them.-  The  organism  sometimes  finds  its 
way  into  milk,  probably  from  carriers  and  lives  there  virulent  for  a  sufficient 
time  to  cause  infection  of  those  who  consume  the  milk. 

Morphology. — The  diphtheria  bacillus  shows  marked  variations  in  size  and 


FIG.  16. — BACILLUS  OF  DIPHTHERIA.       xiooo.      (MacNeal.) 

form;  the  smears  taken  from  the  nose  or  throat  and  from  16  to  20-hour-old  blood- 
serum  cultures  they  usually  appear  as  small  bacilli,  2  to  3  M  long,  0.3  to  0.5  JJL 
broad,  many  are  straight  or  curved,  some  are  club-shaped,  a  few  triangular  and 
some  so  small  as  to  be  coccoid. 

Smears  made  from  cultures  24  to  72  tours  old,  or  older,  show  involution 
forms,  organisms  more  often  club-shaped,  balloon-shaped  or  irregular,  than 
rod-shaped.  Uniform  staining  is  the  exception,  not  the  rule,  especially  among 
involution  forms.  The  corynebacteria,  of  which  the  diphtheria  bacillus  is  a 
type,  when  stained  with  the  ordinary  anilin  stains,  especially  Loeffler's  methy- 
lene  blue,  stain  deeply  at  each  end  and  faintly  in  the  middle,  or  stain  deeply  at 
the  extremities  and  in  the  center,  giving  a  barred  or  granular  appearance. 
When  stained  by  Neisser's  method — acid  methylene  blue,  10  to  15  minutes, 

82 


DIPHTHERIA   BACILLUS  .        83 

washed  in  water  and  Bismarck  brown  applied  for  i  or  2  minutes,  the  bars 
and  granules  appear  deep  blue,  intervening  portions  light  brown.  The  diph- 
theria bacillus  is  Gram  positive.  It  is  not  motile. 

There  are  so  many  variations  in  the  morphology  of  true  diphtheria  bacilli 
and  so  many  similar  pseudodiphtheria  and  non-pathogenic  bacilli  that  it  is 
important  to  know  them.  For  this  reason  and  to  assist  in  the  differentiation 
between  pathogenic  and  non-pathogenic  forms  Westbrook  has  described  a 
classification  which  has  been  accepted  by  many  health  department  laboratories 
and  bacteriologists.  It  may  be  summarized  as  follows: 

Type  A. — Decanter  or  Indian-club  shaped,  showing  one,      i  /*  to  2  /*  thick 
two  or  more  round  granules,  arranged  together,  or  more  fre-      3  ^  to  6  n  long 
quently  distributed;  situated  at  one  or  both  poles,  or  through- 
out the  length  of  the  organism. 

Type  A1. — Same  shape  and  size  as  A  but  presenting  bars  in  place  of  granules. 

Type  A2. — Same  size  and  shape,  solid  uniform  staining. 

Type  B. — Long  slim  straight  or  curved  rods,  one,  two  or        0.5  /z  thick 
more  round  granules  arranged  together,  or  more  frequently        3  p.  to  7  n  long 
distributed;  situated  at  one  or  both  poles  or  throughout  the 
length  of  the  organism. 

Type  B1.- — Same  size  and  shape  as  B  but  presenting  bars  in  pla~°  of  granules. 

Type  B2. — Same  size  and  shape,  solid  uniform  staining. 

Type  C. — Straight  or  curved  rods,  frequently  slightly      0.5  ju  to  i.o  ^  thick 
swollen  at  one  or  both  ends,  one  or  two  round  polar  gran-      3  ^  to  6  /x  long 
ules,  and  occasionally  a  third  granule  near  the  middle. 

Type  C1. — Same  size  and  shape  as  C  but  presenting  bars  in  place  of  granules, 
five  or  more  bars  may  be  observed. 

Type  C2. — Same  size  and  shape,  uniform  staining. 

Type  D. — Straight  or  curved  rods,  two  round  gran-  0.75  JJL  to  i.oo  ju  thick 
ules  situated  one  at  each  pole.  2ju  to  3  p  long 

Type  D1. — Straight  or  slightly  curved  rod  and  carrot-      0.5  y.  to  i.o  M  thick 
shaped,  showing  two,  three,  four  or  occasionally  more       2  ju  to  3  yu  long 
bars. 

Type  D2. — Isosceles  triangle-shaped,  usually  in  pairs  0.75  ju  to  i.oo  ju  thick 
with  bases  in  apposition  solid  and  uniform  staining.  i.o  /-i  to  2.5  n  long 

Type  E. — Straight  or  curved  rods  and  ovoids,  two  0.50  ju  to  0.75  JJL  thick 
round  granules  situated  one  at  each  end.  i  n  to  2  ju  long 

Type  E1.— Same  size  and  shape  as  E  but  presenting  two,  three  or  four  bars 
in  place  of  granules. 

Type  E2. — Same  size,  blunt  cone-shaped,  usually  in  pairs  with  bases  in  appo- 
sition solid  and  uniform  staining. 

Type  F. — Straight  rods,  one  round  polar  granule  and  0.25  ^  to  0.50  /z  thick 
occasionally  a  central  granule.  i  /*  to  2  '/*  long 

Type  F2.— Same  size,  irregular  rod-shaped,  solid  uniform  staining. 

Type  G. — Ovoids,  two  small  round  granules  situ-  0.50  /x  to  0.75  ju  thick 
ated  one  at  each  pole.  i.o  ju  to  1.5  /*  long 


84 


MEDICAL  BACTERIOLOGY 


Type  G2. — Same  shape  and  size  as  G,  solid  uniform  staining.  When  stained 
with  Loeffler's  methylene  blue,  granular  and  barred  forms  of  diphtheria  bacilli 
are  occasionally  polychromatic,  the  granules  and  bars  having  a  reddish  tint. 
The  metachromatic  appearance  is  given  as  a  characteristic  of  Westbrook's  types 
A,  C,  D,  E,  F  and  G. 

Growth. — The  diphtheria  bacillus  is  aerobic  and  grows  at  temperatures  be- 
tween 2o°C.  and  42°C.,  best  at  37°C.  The  most  favorable  media  for  its  culti- 
vation are  veal  broth  and  Loeffler's  blood  serum. 

Bouillon  (preferably  veal  infusion)  incubated  at  37°C.  after  24  to  48  hours 
shows  a  thin  white  film  on  the  surface,  later  the  growth  precipitates  to  the  bottom 
leaving  the  fluid  clear. 

Loeffler's  blood  serum  on  this  medium,  incubated  at  37°C.  round,  elevated 


* 


FIG.  17. — FORMS  OF  B,  DIPTHERI^E  IN  CULTURES  ON  LOFFLER'S  SERUM. 

A,   Characteristic  clubbed  and  irregular  shapes  with  irregular  staining  of  the  cell  contents. 
X  1 100.     B,  Irregular  shapes  with  even  staining.       X  1000.      (After  Park  and  Williams.) 


pin-point,  grayish- white  colonies  appear  in  1 6  to  20  hours;  they  enlarge,  some 
attaining  the  size  of  a  pin  head  or  larger,  others  coalesce,  forming  an  irregular 
outlined  grayish  film  in  24  to  48  hours. 

Some  cultures  have  a  moist  appearance  and  some  have  a  light  yellow  tint. 
Several  days  after  inoculation  the  organisms  show  marked  involution  and  the 
culture  becomes  dry  and  tends  to  die. 

Agar. — On  agar  growth  has  the  same  appearance  as  on  Loeffler's  medium; 
it  occurs  more  slowly,  is  less  abundant,  more  distinctly  white  and  does  not  die 
out  as  soon  as  on  blood  serum. 

Gelatin. — A  few  pin-point  white  colonies  develop  along  a  stab;  on  the  surface, 
occasionally,  small  round  white  colonies  appear.  Gelatin  is  not  liquefied. 

Milk. — Growth  in  this  medium  neither  sours  nor  coagulates  it. 

Spores  are  not  formed.  Indol  is  not  formed.  Acid  is  formed  from  glycerin, 
glucose,  galactose  and  maltose.  Gas  is  not  formed. 

Resistance. — The  diphtheria  bacillus  is  easily  destroyed  by  heat  in  a  moist 
state,  6o°C.  for  15  minutes  kills,  but  when  dried,  especially  when  enclosed  in  a 
false  membrane,  an  exposure  of  i  hour  at  i2o°C.  in  a  hot-air  sterilizer  is  required 


DIPHTHERIA  BACILLUS  85 

to  kill  it.  In  water  it  lives  a  short  time,  and  exposure  to  sunlight  destroys  it 
in  several  weeks.  When  dried  and  protected  from  sunlight  it  survives  in  dust, 
etc.,  for  many  weeks.  Carbolic  acid,  i  per  cent,  solution  kills  the  diphtheria 
bacillus  in  less  than  2  minutes;  it  is  equally  sensitive  to  other  chemical  germicides. 

Toxin. — The  diphtheria  bacillus  produces  a  powerful  extracellular  toxin'. 
Different  strains  of  the  organism  vary  in  this  respect,  some  forming  little  and 
others  much  toxin.  Alkaline  veal  infusion  broth  is  the  best  medium  in  which 
to  obtain  toxin  and  it  is  best  produced  when  incubation  is  maintained  in  a  moist 
atmosphere  between  35°C.  and  37°C.  For  toxin  production  an  abundant 
supply  of  oxygen  (air)  is  required. 

Diphtheria  toxin  is  filterable,  hence  after  the  bacillus  has  produced  toxin 
in  bouillon,  by  filtering  the  culture  through  an  unglazed  porcelain  tube  the  toxin 
is  recovered  in  the  filtrate  free  from  bacteria.  As  small  an  amount  as  J^oo  cc. 
of  such  a  filtrate  often  contains  sufficient  toxin  to  kill  a  guinea-pig. 

Stored  in  a  cool  dark  place  diphtheria  toxin  gradually  deteriorates  in  the 
course  of  months;  heating  above  58°C.  rapidly  attenuates  it,  ioo°C.  destroys 
in  a  short  time. 

The  diphtheria  toxin  is  water-soluble  and  alcohol-insoluble. 

By  injecting  subcutaneously  varying  amounts  of  toxin  into  different  guinea- 
pigs  of  the  same  weight  the  lethal  dose  can  be  determined. 

A  subcutaneous  injection  of  a  very  small  fraction  of  the  lethal  dose  of  anti- 
toxin acts  on  some  animals  in  such  a  way  as  to  increase  resistance  in  5  to  10 
days;  a  second  injection  of  a  quantity  of  toxin  50  to  100  per  cent,  greater  than 
given  at  first  will  then  further  increase  the  animal's  resistance  and  by  continu- 
ing the  treatment,  giving  subcutaneous  injections  of  progressively  larger  doses 
of  toxin  at  intervals  of  about  a  week  until  from  6  to  12  injections  have  been 
administered,  a  high  degree  of  resistance  or  immunity  is  conferred  upon  the 
animal. 

Blood  serum  obtained  from  an  animal  so  immunized  when  mixed  with 
diphtheria  toxin  neutralizes  it,  makes  it  inert,  and  deprives  it  of  disease-producing 
power.  Such  serum  is  known  as  diphtheria  antitoxin  and  is  used  in  the  treat- 
ment of  diphtheria.  Serum  obtained  from  animals  immunized  against  diph- 
theria toxin  does  not  contain  agglutinins  or  amboceptors. 

Agglutination  tests  and  complement  fixation  tests  are  not  available  in  the 
diagnosis  of  diphtheria,  nor  to  differentiate  diphtheria  bacilli  from  pseudo- 
diphtheria  bacilli. 

Pathogenesis. — The  diphtheria  bacillus  has  a  selective  affinity  for  the  mu- 
cous membranes  of  man,  especially  the  mucosa  of  the  upper  air  passages.  This 
organism  may  be  found  upon  the  mucous  membrane  of  the  throat,  tonsils  or 
nose  of  healthy  people  who  have  never  had  the  disease;  this  is  a  common  occur- 
rence among  those  in  contact  with  diphtheria  patients  and  relatively  rare 
among  other  healthy  persons.  Healthy  persons  who  harbor  the  diphtheria 
bacillus,  so  called  carriers,  are  dangerous,  and  may  transmit  the  bacilli  to  others 
in  whom  the  organisms  promptly  cause  disease.  Epidemics  of  diphtheria 
have  been  traced  to  such  sources,  especially  among  school  children. 


86  MEDICAL  BACTERIOLOGY 

After  a  diphtheria  patient  has  entirely  recovered  from  the  disease  he  may 
still  harbor  the  bacillus  for  days,  weeks  or  months. 

In  the  vast  majority  of  cases  the  diphtheria  bacillus  lodges  upon  the  mucous 
membranes  of  the  nose  or  throat,  mostly  on  the  tonsils,  not  infrequently  on  the 
larynx.  If  active  it  causes  a  false  membrane  to  form  on  these  surfaces.  This 
membrane  is  at  first  dirty  white  or  grayish,  as  it  ages  it  becomes  darker.  When 
stripped  off,  the  exposed  mucous  membrane  is  inflamed  and  may  show  bleeding 
points.  Although  the  diphtheria  bacilli  are  confined  to  the  mucous  and  false 
membrane  and  do  not  penetrate  underlying  structures  nor  enter  the  blood-  or 
lymph-streams,  diphtheria  is  a  disease  with  profound  systemic  toxemia.  The 
toxin  liberated  by  the  bacilli  upon  the  mucous  membrane  is  absorbed  and  dis- 
seminated through  the  blood-stream. 

Rare,  isolated  cases  have  been  reported  in  which  the  diphtheria  bacillus  has 
attacked  the  ear,  the  intestine  and  the  genito-urinary  canal. 

Occasionally  the  pus  from  chronic  urethritis  of  long  duration,  when  ex- 
amined microscopically,  shows,  among  other  organisms,  bacilli  morphologically 
indistinguishable  from  diphtheria  bacilli.  Most  of  these  are  not  diphtheria 
bacilli,  some  produce  toxin  in  broth  and  apparently  are  true  diphtheria  bacilli. 
Whether  diphtheria  bacilli  or  not,  they  do  not  cause  false  membrane  forma- 
tion and  do  not  cause  toxemia;  they  are  active  in  maintaining  the  urethritis, 
as  evidenced  by  rapid  recovery  after  they  cease  to  exist  in  the  urethra. 

The  diphtheria  bacillus,  injected  subcutaneously,  is  virulent  for  horses, 
cattle,  dogs,  cats,  rats,  guinea-pigs,  chickens  and  many  other  birds  and  animals. 
Cats  are  said  to  be  subject  to  diphtheria. 

Marked  variations  in  virulence  and  toxin  production  are  exhibited  by  dif- 
ferent strains  of  diphtheria  bacilli.  The  virulence  and  toxin  production  of  any 
diphtheria  bacillus  depends,  to  a  large  extent,  upon  little  understood  condi- 
tions of  environment.  A  diphtheria  bacillus  may  exist  in  one  person's  throat 
for  days  or  weeks  without  injury  to  the  host  and  immediately  cause  disease 
when  transferred  to  another.  A  diphtheria  bacillus  cultivated  in  bouillon  may 
produce  so  little  toxin  that  10  cc.  or  more  of  the  filtrate  is  required  to  kill  a 
guinea-pig,  but  when  the  same  organism  is  transplanted  into  a  different  bouillon, 
one  more  nearly  adapted  to  its  requirements,  so  much  toxin  will  be  produced 
that  o.oi  cc.  or  less  of  the  filtrate  will  kill  a  guinea-pig. 

Some  strains  of  diphtheria  bacilli  are  more  constant  in  their  degree  of  viru- 
lence and  toxin  production  than  others  and  less  susceptible  to  changes  in 
environment. 

Diagnosis. — When  the  diphtheria  bacillus  is  present  in  the  nose,  throat  or 
tonsils,  a  sterile  cotton  swab  passed  over  the  infected  surface  and  then  smeared 
on  a  glass  slide  may  deposit  bacteria  on  the  slide  that  will  be  visible  when 
stained  and  examined  microscopically,  or  it  may  not.  If  after  passing  a  sterile 
cotton  swab  over  the  infected  area,  the  swab  is  then  drawn  across  the  surface 
of  Loeffler's  blood  serum  and  the  medium  incubated  16  to  24  hours  at  37°C., 
any  bacteria  introduced  will  multiply  and  form  colonies  from  which  slides  may 
be  prepared  and  stained  for  microscopic  examination  and  the  organism  ex- 


DIPHTHERIA  BACILLUS  87 

amined.  It  is  desirable  to  establish  the  bacteriological  diagnosis  in  suspected 
cases  of  diphtheria  early  as  possible,  and  for  this  reason  both  culture  and  slides 
should  be  obtained  from  the  patient. 

Occasionally  diphtheria  bacilli  will  be  found  in  the  nose  and  not  in  the  mouth 
or  throat,  hence  slides  and  cultures  should  be  made  from  both  the  nose  and 
throat. 

TECHNIQUE 

Take  two  tubes  of  sterile  Loeffler's  blood-serum  medium,  two  sticks  or  pieces 
of  wire  about  6  inches  long  having  cotton  wrapped  around  one  end  (when  made 
such  swabs  are  placed  in  test-tubes  stoppered  with  cotton,  and  the  tubes  con- 
taining swabs  are  sterilized  at  i4o°C.  for  i  hour  in  hot-air  sterilizer)  and  several 
clean  glass  slides. 

Inspect  the  patient's  mouth  and  throat;  if  grayish- white  spots  or  areas  of 
inflammation  are  observed  rub  the  swab  over  these  and  immediately  draw  across 
the  surface  of  the  Loeffler's  medium;  then  rub  the  swab  on  one  or  more  glass 
slides  and  replace  it  in  its  original  container.  If  no  grayish  spots  or  areas  of 
inflammation  are  observed  draw  the  swab  over  each  tonsil  and  the  pharynx. 

Take  the  second  swab  and  pass  it  into  each  nostril,  then  draw  it  across  the 
surface  of  a  sterile  tube  of  Loeffler's  medium  and  make  slides  from  it;  finally, 
replace  this  swab  in  its  original  container. 

Place  the  blood-serum  culture  tubes  in  incubator  at  37°C.;  stain  the  slides 
for  5  minutes  with  Loeffler's  methylene  blue,  wash  them  in  water,  dry  and 
examine  under  the  microscope.  If  diphtheria  bacilli  are  observed  the  diag- 
nosis is  apparent. 

After  1 6  to  24  hours  remove  cultures  from  incubator,  note  the  macroscopic 
appearance  of  colonies  that  have  developed;  remove  some  of  the  growth  with 
a  sterile  platinum  loop  and  smear  on  slides,  fix  by  gently  heating  slides  until 
dry,  stain  with  Loeffler's  methylene  blue  and  examine  microscopically. 

When  slides  and  cultures  from  a  true  case  of  diphtheria  are  examined  many 
diphtheria  bacilli  are  usually  found,  occasionally  they  are  few.  They  may  be 
found  in  pure  culture — no  other  organism  appearing;  as  a  rule,  however,  staphy- 
lococci,  streptococci  or  pneumococci  are  found  together  with  the  diphtheria 
bacilli. 

'  Usually  virulent  diphtheria  bacilli,  as  observed  in  smears  made  directly 
from  the  nose  and  throat  or  from  16  to  20-hour-old  blood-serum  cultures,  are 
slim,  straight  and  curved  rods  showing  granular  or  barred  staining. 

When  such  smears  show,  almost  entirely,  solid  staining,  short,  stout  rods, 
the  organisms  usually  are  not  virulent,  toxin-producing  bacteria. 

There  are  so  many  exceptions  to  these  rules,  however,  that  occasional  error 
is  inevitable  when  diagnosis  is  based  on  the  appearance  of  bacteria,  and  under 
such  conditions  the  welfare  of  patients  and  public  is  best  served  by  making  a 
diagnosis  of  diphtheria  whenever  smears  or  cultures  obtained  from  a  patient 
having  sore  throat,  fever  and  signs  of  toxemia,  show  organisms  having  the  mor- 
phology of  either  the  diphtheria  or  pseudodiphtheria  bacilli. 


MEDICAL  BACTERIOLOGY 

Shick  Test. — One  of  the  recent  additions  to  diagnostic  tests  has  disclosed 
much  valuable  information  in  regard  to  the  prevalence  of  immunity  and  sus- 
ceptibility of  persons  of  various  ages  in  civilized  communities  and  promises  to 
be  of  practical  value  in  the  future  to  determine  whether  or  not  any  individual 
is  immune  or  susceptible  to  diphtheria. 

The  test  is  based  on  the  findings  of  von  Behring  and  others  that  if  one  has 
as  much  as  J^Q  umt  of  antitoxin  per  cubic  centimeter  in  his  blood  serum  he  is 
immune.  An  amount  of  toxin  which  requires  the  presence  of  J^o  unit  anti- 
toxin per  cubic  centimeter  to  neutralize  it  is  injected  into  the  skin.  If  it  is 
neutralized  inflammation  does  not  occur.  If  not  neutralized,  in  24  to  48  hours, 
an  area  of  erythema  i  to  2  centimeters  in  diameter  appears  with  slight  infiltra- 
tion. Shick  recommends  the  use  of  J^0  unit  of  toxin  in  o.i  cc.  sterile  water. 

The  toxin  should  not  be  diluted  until  the  test  is  to  be  made;  the  injection 
must  be  into  and  not  beneath  the  skin,  if  properly  injected  a  slight  swelling  and 
blanching  occurs  at  the  point  of  injection. 

Strong  or  concentrated  toxin  should  be  employed  so  as  to  minimize  the 
amount  of  protein  from  culture  medium  injected  because  this  is  thought  to  be 
responsible  for  the  pseudoreactions  at  times  observed. 

Carriers. — Persons  who  harbor  diphtheria  bacilli  in  nose  or  throat  without 
suffering  any  injury  therefrom  are  known  as  carriers.  Numerous  investigations 
have  shown  that  most  patients  continue  to  have  diphtheria  bacilli  in  their  nose 
or  throat  for  i  to  2  weeks  after  the  beginning  of  convalescence  from  diphtheria; 
from  0.5  to  5  per  cent,  continue  to  be  carriers  for  weeks  or  months;  and  from 
o.i  to  3  per  cent,  of  persons,  who,  so  far  as  known,  never  had  diphtheria,  harbor 
virulent  bacilli  at  times  or  continuously.  These  carriers  are  most  numerous 
among  nurses  and  physicians  attending  diphtheria  patients. 

Pseudodiphtheria  bacilli  are  occasionally  or  continually  present  in  the  nose 
or  throat  of  from  10  to  80  per  cent,  of  all  people  in  numerous  localities. 

Obviously  it  is  necessary  to  determine  that  a  suspected  or  probable  carrier 
is  free  of  diphtheria  bacilli  before  release  from  quarantine  and  that  organ- 
isms obtained  from  a  suspect  be  tested  for  virulence  as  well  as  examined 
microscopically. 

Kolmer  and  Woody  recommend  the  following  virulence  test  of  organisms 
isolated  from  convalescents  and  suspected  carriers: 

(-4)  i.  Obtain  culture  from  patient  on  Loeffler's  blood  serum.  2.  Trans- 
plant to  0.2  per  cent,  glucose  broth  -f-o.8  reaction.  3.  Incubate  broth  72 
hours.  4.  Inject,  subcutaneously,  an  amount  of  the  culture  equal  to  J£  per 
cent,  the  test  animal's  weight — use  250  to  300  Gm.  guinea-pigs. 

(B)  i.  Procure  a  good  24-hour-old  culture  from  suspected  patient  on  Loeffler's 
medium.  2.  Wash  off  growth  with  10  cc.  of  salt  solution.  3.  Inject  4  cc.  of 
this  salt  solution  suspension  subcutaneously  into  a  250  to  3oo-Gm.  guinea-pig. 

The  test  animal  is  observed  for  4  days  and  if  inflammation  at  point  of  in- 
jection and  toxemia  occur  the  organism  is  considered  virulent. 


CHAPTER  XVIII 
PSEUDODIPHTHERIA  BACILLI 

There  are  a  number  of  organisms,  not  pathogenic,  which  are  similar  to  the 
diphtheria  bacillus  morphologically,  in  staining  and  growth  on  culture  media. 
"They  have  been  found  in  pus,  milk,  urine  and  infectious  processes  resembling 
the  true  diphtheria  bacilli"  (Rosenberger). 

"Differentiation  is  never  difficult"  (Hiss  and  Zinsser).  "Bacillus  diph- 
theriae  forms  acid  from  dextrin,  not  from  saccharose;  Bacillus  xerosis  from 
saccharose  not  from  dextrin;  Bacillus  Hoffmanni  does  not  form  acid  from  either" 
(Hiss  and  Zinsser). 

Hoffman's  bacillus  is  found  in  the  throat  of  healthy  people,  not  infrequently, 
especially  among  school  children  in  poor  districts.  Morphologically  it  is  said 
to  be  shorter  than  the  diphtheria  bacillus  and  more  uniform  in  shape.  It  is 
also  claimed  that  upon  staining  Hoffman's  bacillus  does  not  show  the  irregular 
or  band-like  staining  common  to  the  diphtheria  bacillus  and  when  stained  by 
Neisser's  method  the  entire  bacillus  is  brown,  no  blue  staining  granules  visible. 

Pseudodiphtheria  bacilli  have  a  very  wide  distribution.  They  are  frequently 
found  as  secondary  invaders  in  chronic  suppurative  processes  exposed  to  air. 
They  are  occasionally  discovered  in  soil  and  on  various  utensils.  They  have 
been  regularly  obtained  from  the  infected  tissues  in  Hodgkin's  disease,  and  are 
believed  by  some  to  play  a  part  in  this  disease.  As  to  whether  they  constitute 
the  specific  factor  of  Hodgkin's  disease  is  a  mooted  point;  the  preponderance  of 
evidence  being  against  it. 

Growth. — On  agar,  blood  serum,  gelatin  and  bouillon  the  growth  of  Hoff- 
man's bacillus  is  similar  to  bacillus  diphtheria,  but  more  luxuriant.  In  contrast 
to  the  diphtheria  bacillus  it  does  not  acidulate  media  containing  any  of  the 
sugars. 

Pathogenesis. — Hoffman's  bacillus  does  not  produce  extracellular  toxin, 
does  not  kill  guinea-pigs  nor  rabbits  and  is  not  pathogenic  to  man. 

The  xerosis  bacillus  is  most  often  found  upon  the  conjunctiva,  both  in  health 
and  when  the  eye  is  the  seat  of  disease.  In  morphology  and  staining  it  resembles 
the  diphtheria  bacillus  even  more  closely  than  Hoffman's  bacillus. 

Growth. — The  xerosis  bacillus  may  be  cultivated  on  any  of  the  media  upon 
which  the  diphtheria  bacillus  grows;  its  growth  is  finer,  less  abundant  but 
similar  to  that  of  the  diphtheria  bacillus.  It  forms  acid  on  saccharose,  but  not 
on  dextrin. 

Pathogenesis/ — The  xerosis  bacillus  is  not  pathogenic  for  man,  guinea- 
pigs  nor  rabbits.  It  does  not  form  toxin. 

Diagnosis. — With  few  if  any  exceptions  diphtheria  and  pseudodiphtheria 
must  be  thought  of  and  a  differentiation  made  whenever  an  organism  presum- 

89 


QO  MEDICAL  BACTERIOLOGY 

ably  a  pseudodiphtheria  bacillus  is  found  associated  with  an  infection  of  the 
nose  or  throat.  Experienced  clinical  bacteriologists  can  usually  do  this  by  ob- 
serving the  morphology  and  staining  of  microscopic  preparations  together  with 
the  character  of  growth  on  Loeffler's  blood-serum  medium.  It  is  one  of  the 
most  difficult  problems  that  confronts  the  beginner. 

While  there  is  much  confusion  and  disagreement  on  the  subject  it  seems 
most  probable  that  we  have  to  deal  with  organisms  that  naturally  divide  them- 
selves into  three  groups : 

First,  true  diphtheria  bacilli  which  have  temporarily  lost  some  of  their 
faculties. 

Second,  several  distinct  species — not  diphtheria  bacilli — neither  pathogenic 
or  toxin  producers,  including  Hoffman's  bacillus  and  the  xerosis  bacillus. 

Third,  a  numerous  heterogeneous  group  of  non-pathogenic  saprophytes 
more  or  less  similar  to  and  always  distinguishable  from  typical  diphtheria 
bacilli.  This  third  group  is  almost  ubiquitous. 


CHAPTER  XIX 
THE  BACILLUS  AND  SPIRILLUM  OF  VINCENT 

Vincent's  angina  is  an  infection  of  the  tonsils,  mouth,  pharynx  or  tongue 
caused  by  two  organisms  in  symbiosis — the  bacillus  of  Vincent,  sometimes  called 
fusiform  bacillus,  and  the  spirillum  of  Vincent. 

The  bacillus  of  Vincent,  or  bacilli  indistinguishable  from  it,  are  occasion- 
ally found  in  the  mouth,  not  associated  with  the  spirillum  of  Vincent,  when 
there  is  no  disease. 

The  spirillum  of  Vincent,  or  spirilla  indistinguishable  from  it,  are  occasion- 
ally found  in  the  mouth,  not  associated  with  the  bacillus  of  Vincent,  when  there 
is  no  disease. 

Whenever  both  these  organisms  coexist  in  the  mouth  there  are  always  ob- 
vious signs  of  disease;  the  evidence  indicating  that  it  is  caused  by  this  spirillum 
and  bacillus  and  dependent  upon  their  symbiotic  relation. 

Reported  findings  indicate  that  these  organisms  seldom,  if  ever,  attack  any 
part  of  the  body  other  than  the  mouth  and  pharynx;  that  they  remain  localized 
in  the  superficial  lesion  and  that  their  injurious  effect  is  largely,  probably  en- 
tirely, limited  thereto. 

Morphology. — The  bacillus  of  Vincent  is  fusiform,  spindle-shaped,  or,  more 
often,  shaped  like  a  banana;  it  may  be  perfectly  straight,  but  frequently  is 
slightly  curved.  Typical  organisms  are  thickest  in  the  middle  and  taper  to 
pointed  ends.  The  bacillus  of  Vincent  is  about  5  to  10  /z  long  and  0.6  IJL  wide  in 
the  middle.  It  is  not  motile. 

The  spirillum  of  Vincent  varies  from  5  to  20  y,  in  length,  is  slender  in  propor- 
tion and  shows  irregular  curves — irregular  as  to  number  and  depth — some- 
times they  are  very  distinct  and  again  very  faint.  • 

Staining. — Both  the  bacillus  and  spirillum  of  Vincent  stain  with  the  usual 
anilin  stains,  best  with  carbol  fuchsin,  and  are  Gram  negative. 

Growth. — The  bacillus  and  spirillum  of  Vincent  cannot  be  cultivated  ex- 
cept under  anaerobic  conditions  at  37°C.  Growth  appears  on  serum-agar  and 
LoefBer's  blood  serum  in  2  or  3  days  as  pin-point,  round,  grayish  colonies. 
These  colonies  enlarge  and  may  attain  a  diameter  of  2  millimeters  and  may  show 
a  yellow  tint. 

Many  tubes  of  serum  agar  and  other  media,  regardless  of  how  planted  and 
incubated,  fail  to  show  growth.  Attempt  to  obtain  cultures  are  frequently 
frustrated  by  overgrowth  of  other  organisms  present  in  the  material  taken  from 
lesions. 

Pathogenesis. — The  name  angina  was  given  to  the  infection  caused  by  the 
spirillum  and  bacillus  of  Vincent  because  the  first  cases  studied  presented  the 
superficial  clinical  appearance  of  diphtheria.  Subsequent  observations  have 
proved  this  a  misnomer. 

91 


92  MEDICAL  BACTERIOLOGY 

At  least  three  distinct  clinical  forms  are  observed: 

First,  acute  onset,  beginning  as  stomatitis,  pharyngitis  or  unilateral  tonsil- 
litis, the  inflamed  area  covered  in  several  days  by  a  pseudomembrane  and  pre- 
senting the  appearance  of  diphtheria — after  the  pseudomembrane  is  cast  off, 
the  subsequent  course  may  be  as  in  diphtheria  or  slight  superficial  ulceration  or 
deep  ulceration  may  occur  and  persist  for  several  days  or  weeks — those  cases 
which  at  first  are  clinically  indistinguishable  from  diphtheria  are  about  50  per 
per  cent,  of  the  whole. 

Second,  ulceration,  most  frequently  involving  one  tonsil,  rarely  occurring 


FIG.  1 8. — SPREAD  FROM  A  CASE  OF  VINCENT'S  ANGINA,  SHOWING  SPIRILLA  AND  FUSIFORM 

BACILLI.     STAINED  WITH  METHYLENE  BLUE. 
(4  X  Eyepiece  and  M2  oil  immersion  objective.), 

on  tongue,  occasionally  on  gum,  buccal  mucosa  or  pharynx —  ulcer  develop- 
ing as  do  syphilitic  ulcers,  having  the  punched-out  appearance  and  other  physi- 
cal characteristics  of  a  chancre — frequently  unaffected  by  any  form  of  treatment 
and  persisting  for  weeks  or  months — these  cases  which  always  suggest  syphilis 
constitute  about  40  per  cent,  of  the  whole. 

Third,  insidious  onset,  of  months  or  years  duration  and  marked  by  the 
occurrence,  most  commonly  on  the  gums,  of  one  or  more  vesicles  which  develop 
and  subside  repeatedly — all  of  which  contain  bacilli  and  spirilla,  occasionally 
these  vesicles  ulcerate — these  cases  form  a  very  small  per  cent,  of  the  whole. 

Diagnosis. — In  all  cases  of  pharyngeal,  tonsillar  or  buccal  infection  or  ulcer 
a  sterile  cotton  swab  should  be  passed  over  the  inflamed  area  and  then  rubbed 
on  slides  to  be  fixed,  stained  and  examined  microscopically. 

At  the  same  time  a  tube  of  Loeffler's  medium  should  be  inoculated  and  incu- 
bated aerobically  at  37°C. 

Both  these  procedures  are  advisable  for  the  following  reasons : 

If  the  condition  be  diphtheria,  slides  examined  microscopically,  frequently 


THE  BACILLUS   AND    SPIRILLUM   OF  VINCENT  93 


fail  to  disclose  diphtheria  bacilli,  while  after  incubation  the  cultures  do  reveal 
them. 

If  the  condition  be  Vincent's  angina,  slides  will  always  show  fusiform  bacilli 
and  spiralla;  aerobic  cultures  never  do. 

If,  as  sometimes  occurs,  a  double  infection  exists,  both  Vincent's  angina  and 
diphtheria,  it  is  well  to  recognize  it,  as  such  a  condition  is  said  to  be  more  grave 
than  diphtheria  alone. 

Some  authorities  state  that  slides  made  from  mild  cases  of  Vincent's  angina 
show  relatively  smaller  numbers  than  slides  from  severe  cases.  Usually  the 
organisms  are  quite  numerous.  As  a  rule,  several  or  more  organisms  other  than 
the  spirillum  and  bacillus  of  Vincent  are  found  in  slides  made  from  cases  of 
Vincent's  angina — staphylococci  and  streptococci  most  frequently.  At  times 
the  spirillum  and  bacillus  of  Vincent  are  found  in  practically  pure  culture. 


CHAPTER  XX 
THE  TUBERCLE  BACILLUS 

The  tubercle  bacillus  occurs  in  air,  dust,  soil  and  water  as  a  result  of  con- 
tamination with  feces,  urine,  sputum  or  other  discharges  from  tuberculou- 
animals.  It  is  frequently  present  in  the  milk  of  tuberculous  cows  and  in  prods 
ucts  made  from  it,  such  as  butter  and  cheese.  Muscle  tissue  is  rarely  involved, 
but  tubercle  bacilli  are  numerous  in  the  glands,  lungs  and  viscera  of  diseased 
animals.  Through  contamination  by  dust  and  dirt  and  handling  by  tubercu- 
lous individuals,  various  articles  of  clothing  and  food  are  polluted  with  tubercle 
bacilli. 

It  is  believed  that  bed-bugs  inhabiting  the  bed  of  a  tuberculous  patient 
acquire  tubercle  bacilli,  harbor  them  for  long  periods,  and,  when  opportunity 
occurs,  bite  people  and  in  so  doing,  infect  them. 

This  organism  has  a  wide  distribution  in  every  climate  and  may  enter  man 
through  the  respiratory  tract,  by  inhalation,  through  the  gastro-intestinal 
tract,  by  food,  especially  raw  milk,  possibly  through  the  genital  organs,  by 
sexual  intercourse,  and  through  infection  of  wounds.  In  the  great  majority  of 
cases,  tuberculosis  in  man  follows  infection  through  the  respiratory  tract, 
of  the  rest  most  infections  in  man  are  by  way  of  the  intestinal  tract. 

Morphology. — Tubercle  bacilli  usually  appear  as  small,  straight  or  curved, 
solid-staining  or  beaded  rods,  1.5  to  4.0  M  long  and  0.3  to  0.5  ^  wide.  They  may 
be  arranged  singly  or  in  irregular  clumps.  Organisms  removed  from  old  cul- 
tures are  often  larger  than  those  found  in  tissue  and  frequently  show  branching. 
Beaded  forms  of  tubercle  bacilli  may  look  like  several  small  cocci  in  a  row. 

Staining. — The  peculiar  reaction  of  the  tubercle  bacillus  to  stains,  acids 
and  alcohol  facilitate  the  recognition  of  this  organism  microscopically.  With 
the  exception  of  the  leprosy  bacillus  it  is  probably  the  only  organism  which  is 
both  acid-  and  alcohol-fast.  Presumably  on  account  of  its  fatty  capsule,  the 
usual  anilin  stains  penetrate  it  with  difficulty  or  not  at  all.  When  stained  with 
carbol  fuchsin  neither  acid  nor  alcohol  decolorizes  it  and  other  stains  cannot 
tint  it. 

Of  the  many  methods  of  staining  for  the  tubercle  bacillus  the  following  two 
are  probably  the  best: 

1.  Stain  with  carbol  fuchsin  for  15  minutes  or  longer. 

2.  Immerse  in  Pappenheim's  solution  for  15  minutes,  or  until  the  slide 
looks  blue. 

3.  Wash  in  water. 

4.  Dry  and  examine. 

1.  Stain  with  carbol  fuchsin  for  15  minutes  or  longer. 

2.  Wash  with  5  per  cent,  aqueous  solution  of  nitric  acid  until  bleached 
(i.  e.,  pale  pink). 

94 


THE   TUBERCLE  BACILLUS  95 

3.  Apply  absolute  alcohol  for  30  seconds. 

4.  Wash  in  water  and  apply  methylene  blue  for  5  minutes. 

5.  Wash  in  water. 

6.  Dry  and  examine. 

By  either  of  these  methods  tubercle  bacilli  will  be  stained  red  and  every- 
thing else  blue. 

Growth. — The  tubercle  bacillus  (human  and  bovine  types)  is  an  aerobic 
organism  which  grows  at  temperatures  between  30°C.  and  42°C.,  best  at  37°C. 
It  will  not  grow  on  media  other  than  blood  serum  unless  blood,  blood  serum, 
tissue  or  glycerin  are  added.  Development  is  slow,  growth  first  appears  after 
12  or  14  days'  incubation  and  continues  to  increase  for  3  or  4  weeks. 

Organisms  transplanted  directly  from  the  human  body  or  sputum  to  cul- 
ture media  often  fail  to  grow.  Isolation  of  tubercle  bacilli,  from  other  organ- 
isms by  plating  is  unsatisfactory.  It  is  best  to  inoculate  a  healthy  animal  with 
the  suspected  material  or  material  containing  tubercle  bacilli  or  tubercle  bacilli 
and  other  organisms.  After  3  or  4  weeks  general  tuberculosis  develops  and  if 
the  animal  is  opened  under  aseptic  precautions  and  the  contents  of  enlarged 
tubercular  glands  removed  and  planted  on  appropriate  culture  media,  pure 
cultures  can  be  obtained. 

Glycerin  Bouillon. — To  obtain  growth  on  this  medium  several  large  loops- 
ful  of  an  actively  growing  young  culture  must  be  obtained  and  floated  on  the 
surface  of  the  glycerin  bouillon.  The  glycerin  bouillon  may  have  i  per  cent, 
glucose  added  to  it  with  advantage;  it  must  be  slightly  alkaline  and  have  a  good 
supply  of  oxygen,  obtained  by  placing  the  bouillon  in  a  broad  flask  with  a  wide 
neck.  After  2  to  3  weeks'  incubation  at  37°C.  growth  appears  on  the  surface; 
at  first  a  small,  thin  whitish  scum,  which  rapidly  spreads  over  the  entire  surface 
and  continues  to  grow  thicker  for  a  month  or  more.  The  pellicle  becomes 
wrinkled  or  granular  as  it  ages,  portions  may  fall  to  the  bottom  of  the  flask,  and 
when  the  culture  is  old  the  growth  may  change  from  white  to  yellowish.  The 
medium  remains  clear. 

Glycerin-agar. — Growth  appears  in  about  2  weeks;  at  first  as  dry,  scaly 
white  spots.  These  coalesce,  forming  a  thick,  dry,  corrugated  film,  described 
as  "bread-crumb"  growth. 

Serum-agar. — Growth  is  the  same  as  on  glycerin-agar  except  that  this  is  a 
more  favorable  medium  for  initial  cultivation  after  removal  from  a  guinea-pig. 

Coagulated  Serum. — Growth  is  the  same  as  on  serum-agar. 

The  tubercle  bacillus  does  not  form  spores. 

Resistance. — Tubercle  bacilli  are  most  resistant  when  in  tissue,  least  re- 
sistant when  in  culture  media.  Putrefaction  of  tissue  containing  them  does  not 
lessen  their  vitality  nor  virulence.  To  destroy  tubercle  bacilli  in  tissue,  sputum, 
feces,  etc.,  boiling  for  J£  hour  is  required.  Their  resistance  to  dry  heat  is  so 
great  that  a  temperature  of  i5o°C.  for  several  hours  is  necessary  to  destroy 
them.  Destruction  by  chemical  germicides  is  difficult,  thorough  exposure  to 
strong  solutions  is  necessary. 

The  tubercle  bacillus  resists  drying  and  retains  its  virulence  in  the  dried 


96  MEDICAL  BACTERIOLOGY 

state  for  months,  even  when  exposed  to  sunlight.  In  water  it  survives  longer 
than  in  dust.  Prolonged  cultivation  at  temperatures  above  the  optimum  is 
said  to  lessen  its  virulence  in  time. 

Toxin. — The  tubercle  bacillus  produces  a  powerful  toxin.  There  is  prob- 
ably both  extra-  and  intracellular  toxin  formation.  The  more  virulent  the 
bacillus,  the  more  potent  the  toxin  liberated.  The  intracellular  toxin  of  the 
tubercle  bacillus  known  as  tuberculin  is  resistant  to  heat,  a  temperature  of 
i5o°C.  being  required  to  destroy  it. 

Dead  tubercle  bacilli  injected  into  an  animal  produce  symptoms  and  lesions 
indistinguishable  from  those  produced  by  living  bacilli;  tuberculin,  injected  in 
sufficient  amounts,  has  the  same  effect. 

Tuberculous  subjects  are  very  much  more  sensitive  to  tuberculin  than 
non-tuberculous  subjects,  hence  the  careful  administration  of  tuberculin  serves 
as  a  diagnostic  aid. 

The  employment  of  tuberculin  in  the  treatment  of  tuberculosis  is  of  little, 
if  any,  value  in  the  vast  majority  of  cases.  Its  administration  as  a  therapeutic 
agent  is  a  highly  specialized  art  and  is  fraught  with  danger. 

Attempts  to  immunize  healthy  animals  against  tuberculosis  by  the  injection 
of  tuberculin  have  been  unsuccessful. 

Sera  prepared  to  combat  the  disease  have  failed  to  do  so. 

Tuberculins  produced  by  the  human  tubercle  bacillus,  the  bovine  tubercle 
bacillus  and  the  avian  tubercle  bacillus  are  identical  in  nearly  all  particulars. 
A  man  infected  with  the  human  tubercle  bacillus  is  more  sensitive  to  human 
tuberculin  than  to  bovine  tuberculin;  a  man  infected  with  the  bovine  bacillus 
is  more  sensitive  to  bovine  tuberculin  than  to  human  tuberculin. 

Agglutinins  for  the  tubercle  bacillus  may  be  detected  in  the  blood  and  serous 
exudate  of  some  tuberculous  animals  and  not  in  others;  this,  in  addition  to  the 
difficulty  of  determining  reactions,  precludes  the  use  of  agglutination  tests  for 
diagnosis. 

Complement  fixation  tests  are  not  yet  applicable  to  the  problems  of  diag- 
nosis and  treatment  in  this  disease.  Investigations  have  shown  that  comple- 
ment fixing  bodies  are  present  in  detectable  amount  in  the  blood  serum  of 
patients  suffering  with  active  tuberculosis  and  disappear  when  the  disease  is 
arrested,  but  all  efforts  up  to  the  present  to  produce  a  stable,  reliable  antigen 
have  been  unsuccessful. 

Pathogenesis. — Most  fatal  infections  in  man  are  caused  by  the  tubercle 
bacillus. 

Tuberculosis  may  be  a  purely  localized  disease  involving  only  the  skin,  eye, 
a  bone,  an  articulation,  a  kidney  or  other  organ;  tuberculosis  of  the  lungs  with 
or  without  involvement  of  other  parts  is  by  far  the  most  common  form  of  the 
disease  in  man.  It  may  be  generalized  with  lesions  in  many  and  widely  sepa- 
rated parts  of  the  body,  with  or  without  bacteria  present  in  the  blood. 

In  man  the  majority  of  infections  are  caused  by  the  human  tubercle  bacillus, 
some  are  caused  by  the  bovine  bacillus  and  perhaps  the  avian  bacillus  is  the 
offending  organism  in  rare  cases.  Ichthic  tubercle  bacilli  probably  never 
infect  man. 


FIG.  19. — TUBERCLE  BACILLI  IN  SPUTUM. 

Top  half  illustrates  smear  from  sputum  as  expectorated.  Bottom  half  illustrates  smear 
from  sputum  after  centrifugalizing.  Stained  with  carbol-fuchsin  and  Pappenheim's  solu- 
tion. (4  X  eyepiece  and  Vn  oil  immersion  objective.) 


THE   TUBERCLE   BACILLUS  97 

Tuberculosis,  due  to  the  bovine  tubercle  bacillus  prevails  extensively, 
among  domestic  cattle.  Dogs,  cats,  horses,  sheep  and  goats  occasionally  are 
infected. 

DIAGNOSIS 

Examination  of  Sputum. — The  patient  is  supplied  with  a  clean,  sterile,  wide- 
mouthed  container  for  the  collection  of  the  sample.  That  which  is  expector- 
ated during  the  early  hours  after  arising  from  sleep  is  most  apt  to  contain  tubercle 
bacilli;  hence,  the  patient  is  directed  to  collect  it.  If  expectoration  be  scant 
it  may  be  an  advantage  to  collect  the  output  of  an  entire  day. 

When  received,  the  sputum  is  inspected  and  if  pearl-like  bodies  or  particu- 
larly dense  portions  are  observed  these  are  selected  for  examination,  as  being 
the  most  likely  portions  in  which  to  find  bacteria.  If  the  sputum  is  homo- 
geneous it  should  be  thoroughly  shaken  before  removal  of  a  portion. 

The  selected  particle  of  sputum  is  lifted  with  a  sterile  platinum  loop  and 
placed  upon  a  clean  glass  slide  or  cover  glass;  if  fluid  it  can  be  readily  spread  in 
a  thin,  even  film  with  the  platinum  loop;  if  tenacious  or  granular  an  even  film 
is  obtained  by  placing  a  loopful  of  sputum  on  a  slide,  dropping  a  second  slide 
on  top  of  it  and  then  drawing  the  slides  apart. 

When  the  sputum  has  been  spread  in  a  thin,  even  film  on  the  slide  or  cover 
glass  it  is  fixed  to  the  slide  either  by  letting  it  stand  in  the  room  until  dry,  or 
gently  heating  over  a  flame  until  dry.  After  fixing  by  either  of  these  methods 
it  is  stained  by  one  of  the  methods  devised  to  show  the  acid-  and  alcohol-fast 
properties  of  the  tubercle  bacillus,  previously  described. 

If  examination  of  slides  so  prepared  fails  to  disclose  the  presence  of  tubercle 
bacilli  it  does  not  necessarily  indicate  that  the  sputum  is  free  from  them;  it 
merely  shows  that  if  present  they  are  scant  and  not  found  in  all  portions  of  the 
sputum.  Under  such  circumstances  the  sputum  should  be  treated  as  follows: 

Add  an  equal  volume  of  sterile  water,  shake  until  homogenized,  centrifu- 
galize  at  high  speed  for  Y±  hour  and  make  smears  of  sediment  for  staining  and 
microscopic  examination;  or: 

Add  antiformin  to  the  entire  sample  of  sputum  (i  cc.  of  antiformin  to  4  cc. 
of  sputum),  mix  by  shaking  and  place  in  incubator  for  8  to  24  hours;  centri- 
fuge until  a  precipitate  is  thrown  down,  take  off  the  supernatant  fluid  with  a 
pipette,  replace  it  with  sterile  water,  shake  the  tubes  to  thoroughly  mix  the 
sediment  with  the  water  and  spin  again  until  complete  precipitation;  in  this 
manner  wash  the  precipitate  several  times;  then,  smear  it  on  a  slide  and  fix 
and  stain  the  same  as  sputum. 

Antiformin  dissolves  practically  all  the  sputum  and  all  bacteria  except  the 
tubercle  bacillus;  with  it  we  can  concentrate  all  the  tubercle  bacilli  that  might 
be  present  in  50  cc.  of  sputum  into  a  precipitate  of  less  than  i  cc. 

It  is  to  be  remembered  that  a  tuberculous  person  may  only  occasionally 
expectorate  sputum  containing  tubercle  bacilli.  Failure  to  find  tubercle  bacilli 
in  sputum  from  a  suspected  patient  should  not  be  considered  significant  until 
samples  have  been  obtained  and  examined  on  several  consecutive  days. 

7 


QO  MEDICAL  BACTERIOLOGY 

If  concentration  and  repeated  microscopic  examination  of  sputum  fail  to 
reveal  bacilli  in  a  suspected  case,  a  recently  obtained  sample  of  sputum  should 
be  emulsified  with  sterile  water  and  injected  subcutaneously  or  into  the  peri- 
toneal cavity  of  a  guinea-pig.  If  tuberculosis  .is  caused  by  such  an  injection 
it  becomes  apparent  in  4  to  6  weeks.  After  such  time  has  elapsed  the  animal 
is  opened  and  examined  for  tubercular  lesions. 

Examination  of  Spinal  Fluid. — Obtain  from  10  to  20  cc.  of  spinal  fluid  in 
a  sterile  test-tube.  If  the  fluid  is  clear  and  has  the  gross  appearance  of  normal 
fluid,  avoid  agitation,  place  in  ice  box  over  night  and  inspect.  The  formation 
of  a  delicate  cobweb-like  coagulum  suspended  in  the  fluid  is  indicative  of 
tuberculous  meningitis.*  This  phenomenon  occurs  only  when  the  fluid  is  pro- 
tected from  agitation — i.e.,  transported  but  a  short  distance  from  the  patient 
to  an  ice  box,  and  that  without  shaking.  The  tube  must  not  be  disturbed  while 
in  the  box,  and  be  removed  with  care. 

Spinal  fluid  may  also  be  examined  for  tubercle  bacilli  by  centrifugalizing 
10  cc.  or  more  of  it  and  making  smears  of  the  sediment  on  slides,  staining  for 
tubercle  bacilli  and  examining. 

Examination  of  Urine. — A  sterile  bottle  containing  a  crystal  of  thymol  is 
used  to  collect  the  urine. 

The  external  genitalia  should  be  washed  to  remove  smega  and  other  bacilli 
before  obtaining  the  urine,  whether  it  is  obtained  by  catheter  or  micturition. 

Repeated  examination  may  be  necessary  before  tubercle  bacilli  are  dis- 
covered. 

Morning  urine  or  the  output  of  an  entire  day  may  be  taken  for  examination. 

It  may  be  centrifugalized  in  sterile  tubes  and  the  entire  sediment  collected 
and  washed  with  sterile  water  once  or  twice  and  then  spread  on  slides,  dried, 
stained  and  examined,  or  the  sample  may  be  treated  with  antif ormin  as  follows : 

Place  the  urine  in  a  conical  glass  and  add  antiformin,  a  few  drops  at  a  time 
until  a  light  precipitate  begins  to  fall.  Cover  the  vessel  and  put  in  a  warm4 
place  or  leave  at  room  temperature  over  night.  Syphon  off  the  supernatant 
fluid  and  collect  the  sediment  in  sterile  centrifuge  tubes,  wash  two  or  three  times 
with  sterile  water,  spread  on  slides,  dry,  stain  and  examine. 

If  a  sample  of  6  ounces  or  less  of  urine  is  to  be  examined  it  is  best  to  sediment 
and  wash  with  water  and  not  use  antiformin.  Antiformin  is  of  most  value 
when  the  sediment  from  a  large  quantity  is  to  be  examined. 

When  microscopic  examinations  fail  to  disclose  tubercle  bacilli  and  guinea- 
pig  inoculations  are  desirable,  the  sediment  from  10  to  30  cc.  of  urine  should  be 
mixed  with  water  and  several  cubic  centimeters  injected  into  the  peritoneal 
cavity. 

Examination  of  Blood. — Under  strict  aseptic  precautions  obtain  from  10 
to  50  cc.  of  blood  from  a  vein  with  syringe  and  needle,  add  a  50  per  cent,  anti- 
formin solution,  a  little  at  a  time  and  keep  in  incubator  until  the  blood  is  entirely 

*  Very  rarely  diseases  of  the  spinal  cord  or  meninges,  other  than  to  tuberculosis  show  this 
coagulum.     • 


THE   TUBERCLE  BACILLUS  99 

destroyed,  centrifugalize  and  wash  the  sediment  with  sterile  water,  spread  on 
slides,  stain  and  mount. 

Examination  of  pleural  and  other  effusions  and  pus:  centrifugalize  and  make 
spreads  from  sediment,  dry,  stain  and  examine  or  inject  the  entire  sediment 
into  the  peritoneal  cavity  of  a  guinea-pig. 

Microscopic  examination  of  these  fluids  seldom  reveals  tubercle  bacilli 
even  when  the  tubercle  bacillus  is  the  cause  of  their  formation.  When  injected 
into  guinea-pigs  50  per  cent,  or  more  fail  to  produce  tuberculosis. 

Examination  of  Milk. — Centrifugalize  i  pint  until  cream  separates.  Remove 
the  cream,  dissolve  it  in  ether,  centrifugalize  the  ether,  wash  the  sediment  in 
sterile  water  and  inject  it  into  a  guinea-pig,  intraperitoneally. 

Centrifugalize  the  skimmed  milk  until  sedimentation  is  complete  and  inject 
the  sediment  into  a  second  guinea-pig. 

Examination  of  Feces. — Give  the  patient  a  dose  of  salts  the  night  before 
sample  is  collected.  Add  antiformin  to  the  stool  and  allow  it  to  digest  for  12 
hours,  collect  and  wash  the  sediment,  inject  a  couple  of  cubic  centimeters  sub- 
cutaneously  into  a  guinea-pig  and  make  smears  from  the  sediment  for  micro- 
scopic examination. 

DIFFERENTIATION  OF 

Human,  Bovine  and  Avian  types  of  tubercle  bacilli.  Recent  disclosures  sug- 
gest that  when  microscopic  examinations  of  sputum,  urine,  feces,  milk,  etc.,  fail 
to  show  tubercle  bacilli  in  suspected  cases,  cultural  tests  may  do  so  and  it  now 
seems  advisable  to  make  such  tests.  The  author  has  had  most  promising  re- 
sults with  the  methods  recommended  by  Petroff,  with  both  PetrofTs  medium 
and  (William's  and  Burdick's)  modification.  They  all  show  practically  the 
same  staining  characteristics. 

PetrofFs  Method. — Mix  equal  parts  of  sputum  and  3  per  cent,  sodium  hydrox- 
ide solution  and  incubate  at  37°C.  for  J^  hour.  Add  normal  HC1  until  neutral 
to  litmus  paper  (litmus  paper  used  should  be  sterile).  Centrifugalize  at  high 
speed  for  15  minutes.  Pipette  off  supernatant  fluid  and  plant  sediment  on 
Petroff's  medium  or  (William's  and  Burdick's)  rnodification.  Incubate  tubes  at 
37°C.  If  growth  does  not  appear  in  2  weeks  examine  microscopically  as  it  is  fre- 
quently present  but  invisible  from  the  fifth  to  the  fifteenth  day. 

Observed  in  tissue  the  bovine  bacillus  is  shorter  and  thicker  and  more  uniform 
in  size  and  staining  than  the  human;  the  avian  longer  and  relatively  more 
slender,  but  these  differences  in  size  and  shape  are  usually  so  modified  by 
cultivation  on  artificial  media  as  to  make  microscopic  differentiation  frequently 
uncertain  or  impossible. 

The  human,  type  grows  more  rapidly  and  luxuriantly  on  culture  media  than 
the  bovine.  In  acid  glycerin  bouillon  the  human  type  causes  slight  if  any 
reduction  of  the  acidity  during  the  early  weeks  of  cultivation,  later  it  makes 
the  medium  more  acid;  the  bovine  type  reduces  the  acid  reaction  to  or  near 
the  neutral  point  and  does  not  increase  acidity  later. 


IOO  MEDICAL  BACTERIOLOGY 

The  avian  type  develops  best  at  temperatures  too  high  for  the  cultivation  of 
the  other  types. 

The  bovine  type  is  more  virulent  for  guinea-pigs  and  rabbits  than  the  human. 
It  kills  rabbits  in  4  to  6  .weeks,  while  the  human  type  requires  several  months  or 
more  to  kill  rabbits  and  frequently  fails,  too.  The  avian  type  is  only  slightly 
pathogenic  for  guinea-pigs,  and  is  virulent  for  rabbits.  The  ichthic  grows  best 
at  room  temperature  and  is  not  pathogenic  for  Laboratory  animals. 


CHAPTER  XXI 

THE  BACILLUS  OF  LEPROSY,  THE  SMEGMA  AND  OTHER  ACID-FAST 

BACTERIA 

The  bacillus  of  leprosy,  so  far  as  known,  infects  man  only.  This  is  purely  a 
parasitic  organism  that  has  not  been  successfully  cultivated  outside  the  human 
body.  It  is  probable  that  the  leprosy  bacillus,  deposited  in  the  sputum,  nasal 
secretions,  feces  and  discharges  from  lesions  of  the  disease,  on  clothing,  utensils, 
etc.,  may  survive  and  retain  its  infectiousness  for  some  time. 

The  leprosy  bacillus  is  usually  present  upon  the  mucosa  of  the  nares  from 
the  early  stages  and  throughout  the  disease.  It  is  present  in  the  enlarged  glands, 
some  of  the  nodules  and  in  the  ulcerative  lesions  of  the  disease. 

When  the  lungs  are  involved,  sputum  may  contain  them,  and  when  the 
intestines  are  affected  leprosy  bacilli  may  be  found  in  the  feces. 

Diagnosis. — Bacteriological  diagnosis  is  based  upon  microscopic  examina- 
tions. In  every  case  a  sterile  cotton  swab  should  be  rubbed  over  the  nasal 
mucous  membrane  and  then  rubbed  on  a  clean  slide.  If  ulcers  exist  smears  are 
made  from  the  necrotic  surface  and  small  pieces  of  th,e  base  of  the  ulcer  removed 
for  microscopic  examination.  If  ulceration  has  not  occurred,  nodules  and  en- 
larged glands  are  sought  for,  massaged,  and  fluid  extracted  from  them  and  spread 
on  slides. 

After  drying,  the  slides  are  stained  exactly  as  for  tubercle  bacilli,  the  leprosy 
bacillus  having  the  same  morphology  and  staining  characteristics  as  the  tubercle 
bacillus.  The  lesions  of  leprosy  contain  very  many  leprosy  bacilli,  some  of  the 
tissue  cells  containing  large  numbers  of  them;  this  is  a  differential  point  between 
leprosy  and  tuberculosis. 

When  clinical  signs  and  bacteriological  findings  furnish  inadequate  evidence, 
X-ray  examinations  of  the  bones  of  the  feet  and  hands  frequently  show  char- 
acteristic changes. 

THE  SMEGMA  BACILLUS 

The  smegma  bacillus  is  frequently  present  upon  the  external  genitalia  of  both 
men  and  women,  occasionally  on  the  adjacent  cutaneous  surfaces  and  rarely  in 
the  mouth  or  on  the  tonsils.  It  is  a  non-pathogenic  saprophyte  similar  to  the 
tubercle  bacillus  in  many  respects  and  differentiated  from  it  by  the  following: 
(i)  The  smegma  bacillus  is  acid-fast,  but  not  alcohol-fast;  Pappenheim's  SO!UT 
tion,  after  carbol  fuchsin,  decolorizes  it  in  less  than  20  minutes.  (2)  The  smegma 
bacillus  does  not  produce  tuberculosis  when  inoculated  into  guinea-pigs.  (3)  The 
smegma  bacillus  does  not  produce  tuberculin. 

There  are  numerous  acid-fast  bacteria,  including  Rabinowitsch's  butter 
bacillus  and  Moeller's  grass  bacilli,  morphologically  indistinguishable  from  the 
tubercle  bacillus,  which  occur  in  air,  soil,  water,  milk,  butter  and  cheese.  None 


102  MEDICAL  BACTERIOLOGY 

of  these  saprophytes  are  pathogenic  to  man.     Some  are  pathogenic  to  laboratory 
animals.     They  are  differentiated  from  the  tubercle  bacillus  as  follows: 

1.  When  stained  with  carbol  fuchsin  and  treated  with  Pappenheim's  solution 
for  20  minutes  they  appear  blue. 

2.  They  do  not  produce  tuberculin. 

3.  Many  of  them  grow  on  plain  bouillon,  agar,  and  gelatin  and  at  room  tem- 
perature.    (Their  growth,  in  many  respects,  resembles  that  of  the  tubercle 
bacillus.) 

There  has  been  considerable  discussion  and  disagreement  as  to  the  possibility 
of  differentiating  the  tubercle  from  non-pathogenic  acid-fast  bacilli  by  methods 
of  staining,  which  should  stimulate  consideration  of  the  following  facts : 

First,  the  vast  majority  of  tubercle  bacilli,  found  in  tissue,  tuberculous  exu- 
dates  and  pus,  or  directly  cultured  from  them,  when  stained  with  carbol  fuchsin 
cannot  be  decolorized  with  acids  and  alcohol  within  an  hour. 

Second,  occasionally,  tubercle  bacilli  found  in  tissue,  tuberculous  exudates 
pus,  or  in  cultures,  are  decolorized  in  a  few  seconds  or  minutes  with  acid  and 
alcohol  or  with  acid  alone,  after  staining  with  carbol  fuchsin. 

Third,  a  very  large  majority  of  the  non-pathogenic  acid-fast  bacteria,  no 
matter  where  found,  are  decolorized  in  a  few  minutes  with  alcohol,  after  staining 
with  carbol  fuchsin. 

Fourth,  occasionally  non-pathogenic  acid-fast  bacteria,  from  various  sources, 
are  found  which  resist  decolorization  by  alcohol  as  long  or  longer  than  the 
majority  of  tubercle  bacilli — for  hours. 


CHAPTER  XXII 
THE  COLON  BACILLUS 

(BACILLUS  COLI)  » 

The  colon  bacillus  is  a  normal  inhabitant  of  the  intestinal  canal  of  man  and 
many  animals,  including  all  the  domestic  animals.  A  large  portion  of  the  entire 
bulk  of  f eces  is  composed  of  colon  bacilli.  The  colon  bacillus  has  been  found  in 
abundance  upon  plants  and  in  water  of  regions  barren  of  animal  life. 

The  colon  bacillus  is  commonly  present  in  air,  dust  and  soil  and  in  water  pol- 
luted with  sewage.  From  these  sources  contamination  of  milk  and  foodstuffs 
often  occurs. 


FIG.  20. — BACILLUS  COLI.      STAINED  WITH  METHYLENE  BLUE. 
(4  X  eyepiece  and  Ma  oil  immersion  objective.) 

Morphology. — The  colon  bacillus  is  a  straight,  round-end  bacillus,  2  to  3  /* 
long  and  0.5  to  0.8  p  wide.  It  has  from  four  to  eight  lateral  flagella,  which  are 
about  4  /x  long,  and  is  actively  motile.  Colon  bacilli  are  arranged  irregularly,  in 
groups,  singly,  sometimes  in  pairs,  end  to  end,  and  in  long  filaments. 

Staining. — The  colon  bacillus  stains  readily  with  all  the  usual  anilin  dyes 
and  is  Gram  negative. 

Growth. — The  colon  bacillus  is  aerobic  and  to  a  degree  anaerobic.  It  grows 
at  temperatures  between  4°C.  and  46°C.,  best  at  37°C. 

Plain  Bouillon  incubated  at  37°C.  begins  to  show  cloudiness  in  about  12 
hours.  A  light,  whitish  sediment  forms  in  24  to  48  hours,  which  increases  in 
amount  for  several  days.  Eventually,  if  undisturbed,  the  entire  growth  pre- 
cipitates, leaving  the  fluid  clear. 

103 


104  MEDICAL  BACTERIOLOGY 

Plain  Agar. — In  18  to  24  hours,  small,  round,  moist,  whitish-gray  colonies 
appear;  these  coalesce  forming  a  thin  grayish  opaque  film. 

Glycerin  Agar. — On  glycerin  agar  the  growth  has  the  same  appearance,  but 
is  slightly  more  abundant. 

Loeffler's  Blood  Serum. — On  Loeffler's  blood  serum  growth  is  the  same  as  on 
agar. 

Gelatin. — Surface  cultures  are  the  same  as  on  agar.  Gelatin  stab  cultures 
show  a  continuous  yellowish-white  growth  along  the  entire  length  of  the  stab  and 
on  the  surface. 

Gelatin  is  not  liquefied. 

Potato. — On  the  surface  of  potato  slants  a  moist,  yellowish  film  appears  in  24 
to  48  hours;  as  it  ages  it  becomes  thicker  and  darker,  eventually  brown. 

Milk  is  acidulated  and  coagulated  in  from  12  to  48  hours. 

The  colon  bacillus  produces  indol.     It  does  not  form  spores. 

The  colon  bacillus  ferments  glucose,  lactose,  saccharose,  maltose  and  levu- 
lose,  with  both  gas  and  acid  production. 

Resistance. — The  colon  bacillus  is  not  destroyed  by  freezing,  boiling  kills  it 
instantly,  moist  heat  at  6o°C.  kills  it  in  half  an  hour.  It  is  destroyed  in  the  hot- 
air  sterilizer  in  less  than  an  hour  at  ioo°C.  Dry,  it  survives  for  half  a  year  or 
more.  Exposure  to  direct  sun  rays  kills  the  colon  bacillus  in  from  4  to  30  hours. 
In  feces,  soil  and  water,  under  favorable  conditions,  colon  bacilli  remain  alive  for 
many  months.  Resistance  to  chemical  germicides  is  not  great;  a  2  per  cent, 
phenol  solution  will  kill  the  colon  bacillus  in  several  minutes. 

Toxin. — The  colon  bacillus  produces  an  intracellular  toxin.  Filtrates  from 
sterilized  heated  cultures,  injected  into  animals  give  rise  to  the  same  toxic 
symptoms  and  signs  as  infection  with  living  bacilli. 

Agglutinins  occur  in  the  blood  of  infected  persons  and  in  the  blood  of  animals 
immunized  against  the  colon  bacillus.  These  agglutinins  are  not  only  specific 
for  the  colon  bacillus,  but  show  an  especially  strong  action  on  the  particular 
strain  of  the  organism  responsible  for  their  production. 

Lysins  and  Precipitins  also  occur  in  the  blood  of  animals  immunized  against 
the  colon  bacillus. 

Pathogenesis. — The  fact  that  colon  bacilli  in  enormous  numbers  are  con- 
stant inhabitants  of  the  intestinal  canal  would  seem  to  indicate  a  lack  of  patho- 
genic power  of  the  bacillus  or  a  gradual  acquirement  of  immunity  on  the  part  of 
the  host.  There  are  marked  variations  in  virulence,  some  strains  of  colon 
bacilli  being  sufficiently  virulent  to  cause  septicemia  and  death.  As  a  rule,  they 
are  but  slightly  pathogenic. 

In  health  these  organisms  are  confined  to  the  intestinal  canal,  but  when  dis- 
ease or  injury  reduces  the  vitality  of  the  intestinal  wall,  peritoneum,  or  any  organ 
adjacent  to  the  intestines,  colon  bacilli  are  prone  to  migrate  and  attack  the  im- 
paired organ;  thus,  they  are  often  found  infecting  an  appendix,  gall-bladder,  peri- 
toneum, fallopian  tube  or  urinary  bladder. 

Through  causes,  and  by  no  means  not  well  understood,  colon  bacilli  migrate 
to  and  are  often  the  cause  of  renal  infections,  acute,  chronic  or  suppurative. 


THE   COLON  BACILLUS  10$ 

Rarely  they  enter  the  blood-stream  and  produce  a  septicemic  condition  similar 
to  typhoid  fever  and  with  similar  sequelae.  Colon  bacillus  infections  may  not 
all  come  from  within.  This  organism  can,  to  a  certain  degree,  produce  pus. 

Foodstuffs  contaminated  with  colon  bacilli  are  sometimes  fermented  and 
split  into  simpler  compounds,  some  of  which  are  highly  poisonous. 

When  injected  into  the  peritoneal  cavity  of  a  guinea-pig  or  rabbit,  virulent 
colon  bacilli  cause  peritonitis,  septicemia  and  death. 

Diagnosis. — Isolation  and  identification  of  the  colon  bacillus,  and  differentia- 
tion from  the  typhoid  and  paratyphoid  bacilli  will  be  described  in  a  chapter  de- 
voted entirely  to  such  procedures. 


CHAPTER  XXIII 

THE  TYPHOID  BACILLUS 

(THE  EBERTH  GAFFKY  BACILLUS) 

The  typhoid  bacillus  is  present  in  the  blood,  urine  and  feces,  sometimes  in  all 
three,  of  typhoid  fever  patients.  It  is  occasionally  present  in  the  urine  or  feces 
for  weeks,  months  or  years  after  recovery  from  typhoid  fever.  It  is  variously 
stated  by  different  observers  that  from  i  in  500  to  i  in  5000  of  the  inhabitants 
of  a  community  are  typhoid  carriers  and  that  these  are  responsible  for  from  20 
to  50  per  cent,  of  all  typhoid  infections.  The  typhoid  bacillus  may  be  found  in 
ulcers,  abscesses  and  other  complications  or  sequelae  of  typhoid  fever. 

From  such  sources,  especially  feces,  the  organism  may  be  transmitted  to 
soil,  water,  milk,  cooking  and  serving  utensils,  and  various  articles  of  food. 

Morphology. — The  typhoid  bacillus  is  2  to  3  /*  by  0.5  to  0.8  ju,  in  size  is 
straight,  with  rounded  ends,  and  has  from  12  to  24  lateral  flagella  which  are 
about  8  fjL  long.  It  is  actively  motile.  The  typhoid  bacillus  has  been  described 
as  differing  from  the  colon  bacillus  in  the  number  of  its  flagella  and  in  degree  of 
motility.  These  differences  are  so  variable  and  slight  that  differentiation  can- 
not be  based  on  them. 

Staining. — The  typhoid  bacillus  stains  with  all  the  common  stains  and  is 
Gram  negative. 

Growth. — Culture  of  the  typhoid  bacillus  is  subject  to  the  same  conditions 
as  the  colon  bacillus.  Growth  of  the  typhoid  bacillus  on  plain  bouillon,  agar 
and  gelatin  is  indistinguishable  from  that  of  the  colon  bacillus. 

Milk  is  not  coagulated  and  is  not  acidulated. 

Potato. — A  typical  growth  of  the  typhoid  bacillus  on  potato  occurs  in  24 
to  28  hours.  It  is  very  scant,  colorless  and  moist,  almost  or  quite  invisible. 
This  typical  development  does  not  occur  always;  there  may  be  no  growth  or 
the  culture  may  have  the  same  appearance  as  a  colon  culture. 

Indol  is  not  produced.  There  is  no  spore  formation.  On  media  containing 
any  of  the  sugars  the  typhoid  bacillus  does  not  form  gas.  Acidulation  occurs 
with  glucose  but  not  with  lactose  nor  saccharose. 

Resistance. — The  typhoid  bacillus  has  the  same  power  of  resistance  as  the 
colon  bacillus  to  heat,  cold,  drying,  exposure  to  sunlight  and  chemical  germi- 
cides. In  water  saprophytic  bacteria  tend  to  destroy  typhoid  bacilli  so  that 
they  usually  survive  but  a  few  days.  Cases  have  been  reported  in  which 
typhoid  bacilli  were  found  alive  and  virulent  in  water  after  30  days.  In  ice 
they  remain  alive  and  virulent  for  months;  also  in  soil  under  certain  conditions. 

Toxin. — The  typhoid  bacillus  produces  an  intracellular  toxin. 

Agglutinins  occur  in  the  blood  of  nearly  all  typhoid  patients  after  the  first 
week  of  the  disease  and  may  be  found  in  the  blood  of  some  people  weeks,  months 

106 


THE   TYPHOID  BACILLUS 


107 


or  years  after  recovery  from  typhoid  fever.     These  agglutinins  are  specific 
for  the  typhoid  bacillus  and  their  detection  forms  an  important  diagnostic  test. 


FIG.    21. — TYPHOID  BACILLUS. 

Upper  half  of  field  showing  clumps.     Lower  half  of  field  showing  bacilli  free  from  clumps 

after  filtered  through  paper. 


FIG.  22. — TYPHOID  BACILLUS. 

A.  Agar  culture,  showing  a  few  short  filaments.     B.  Broth  culture.     Filaments  much 
longer  and  more  numerous.     (12  X  eyepiece  and  Ma  oil  immersion  objective.) 

Specific  precipitins,  agglutinins  and  lysins  occur  in  the  serum  of  animals  im- 
munized against  the  typhoid  bacillus  by  inoculation  with  sterilized  bacilli. 

Pathogenesis. — The  typhoid  bacillus  is  the  specific  cause  of  typhoid  fever,  a 
septicemia  with  localized  lesions  in  the  small  intestine. 


I08  MEDICAL  BACTERIOLOGY 

During  the  course  of  typhoid  fever  bacilli  lodge  in  the  spleen,  bone-marrow, 
gall-bladder  and  occasionally  in  other  parts;  some  may  survive  after  subsidence 
of  the  fever  and  remain  dormant  for  months  or  years  and  then  cause  an  acute, 
localized,  inflammatory  or  suppurative  condition. 

Occasionally  the  typhoid  bacillus  has  been  found  the  exciting  cause  of  chole- 
cystitis, meningitis  and  pneumonia  without  the  previous  occurrence  of  typhoid 
fever. 

Apparently,  typhoid  fever  and  typhoid  infections  occur  only  in  man.  When 
virulent  typhoid  bacilli  are  injected  into  lower  animals,  septicemia  and  death 
follow  without  the  manifestations  of  typhoid  fever  observed  in  man. 


CHAPTER  XXIV 

• 

THE  PARATYPHOID  AND  SIMILAR  BACILLI 

There  are  a  number  of  organisms  that  produce  manifestations  of  disease  sim- 
ilar to  typhoid  fever.  Some  of  them  closely  resemble  the  typhoid  bacillus  in 
morphology,  staining  and  cultural  characteristics.  Of  these  the  most  important 
are  as  follows: 

PARATYPHOID  A  BACILLUS. 
PARATYPHOID  B  BACILLUS. 
BACILLUS  ENTERITIDIS  OF  GAERTNER. 

PARATYPHOID  A  BACILLUS 

The  paratyphoid  A  bacillus  produces  a  clinical  picture  indistinguishable 
from  a  mild  typhoid  infection  or  typhoid  fever. 

The  distribution  of  the  paratyphoid  A  bacillus  in  the  human  body  is  similar  to 
that  of  the  typhoid  bacillus.  The  resistance  of  these  two  organisms  is  practi- 
cally identical.  Pollution  of  soil,  water,  etc.,  and  the  mode  of  infection  very 
probably  is  the  same  in  paratyphoid  fever  as  in  typhoid  fever. 

The  morphology,  staining,  and  motility  of  the  paratyphoid  A  bacillus  is 
indistinguishable  from  that  of  the  typhoid  bacillus.  The  growth  of  these  two 
organisms  is  the  same  on  most  culture  media. 

Milk  is  not  coagulated  but  is  acidulated  in  from  24  to  48  hours;  the  reaction 
remaining  constantly  acid  thereafter.  In  media  containing  glucose  acid  and  gas 
is  formed.  There  is  no  gas  nor  acid  production  with  lactose. 

Toxin. — The  paratyphoid  A  bacillus  forms  an  intracellular  toxin. 

Agglutinins  are  found  in  the  serum  of  patients  and  inoculated  animals  under 
the  same  conditions  as  in  typhoid  infections.  Just  as  typhoid  agglutinins  are 
specific  for  the  typhoid  bacillus  and  do  not  agglutinate  paratyphoid  bacilli,  so, 
too,  paratyphoid  A  serum  is  specific  for  the  paratyphoid  A  bacillus  and  does  not 
agglutinate  the  typhoid  bacillus.  The  specificity  of,  these  agglutinations  is 
limited  by  the  occurrence  of  group-agglutination  reactions  as  will  be  explained 
in  the  chapter  on  diagnosis. 

PARATYPHOID  B  BACILLUS 

The  paratyphoid  B  bacillus  has  the  same  morphology,  staining  and  resistance 
and,  in  many  respects,  the  same  cultural  characteristics  as  the  typhoid  and 
paratyphoid  A  bacilli. 

Unlike  them,  it  infects  some  of  the  lower  animals,  mice,  swine  and  cattle,  as 
well  as  man.  It  would  seem  that  infections  with  paratyphoid  B  bacillus  more 
often  result  from  the  consumption  of  diseased  and  contaminated  foodstuffs, 
especially  meats,  than  is  the  case  in  typhoid  and  paratyphoid  A  infections. 

109 


110  MEDICAL  BACTERIOLOGY 

The  clinical  manifestations  of  paratyphoid  B  infections  vary.  In  half  or 
more  of  the  cases  the  disease  runs  a  course  similar  to  typhoid  fever.  Frequently 
the  picture  is  that  of  a  gastro-enteritis  of  sudden  onset,  sometimes  indis- 
tinguishable from  true  cholera.  Rarely  the  disease  has  occurred  as  an  acute 
pneumonia. 

The  organism  may  be  found  in  the  blood,  urine  or  feces  of  infected  persons 
and  the  organism  differentiated  from  other  members  of  the  typhoid  colon  group 
by  cultural,  agglutination  and  complement  fixation  tests. 

Milk  is  not  coagulated  by  the  paratyphoid  B  bacillus;  it  is  acidulated  during 
the  first  24  to  48  hours;  in  about  10  days  the  reaction  becomes  alkaline  and 
remains  so  thereafter. 

Glucose  Media  show  acid  and  gas  production;  there  is  no  acid  production  on 
lactose  or  saccharose. 

BACILLUS  ENTERITIDIS  OF  GAERTNER 

The  bacillus  enteritidis  of  Gaertner  has  been  found  in  diseased  mice,  guinea- 
pigs,  rabbits  and  cattle.  Infections  in  man  have  occurred  as  a  result  of  eating 
the  meat  of  diseased  cattle.  This  organism  so  closely  resembles  the  para- 
typhoid B  bacillus  that  careful  agglutination  tests  are  required  to  differentiate 
them.  f 

BACILLUS  ENTERITIDIS  AERTRYCKE 

Bacillus  enteritidis  aertrycke  is  closely  related  to,  if  not  identically  the  same, 
as  paratyphoid  B  bacillus. 

OTHER  MEMBERS  OF  THE  TYPHOID-COLON  GROUP 

From  time  to  time  organisms  are  found  which  have  the  same  distribution  and 
pathogenic  properties  as  the  colon  bacillus;  organisms  that  have  also  the  same 
morphology,  motility  and  staining  characteristics  as  the  colon  bacillus,  and 
apparently  the  same  growth  on  plain  agar,  gelatin  and  bouillon,  but  differ  from 
the  colon,  the  typhoid  and  other  bacilli  in  their  action  on  carbohydrates  and  their 
behavior  when  brought  into  contact  with  sera  containing  specific  precipitins, 
agglutinins  and  lysins. 

Some  of  these  very  probably  are  distinct  species  and  others,  undoubtedly,  are 
atypical  strains  of  colon  or  similar  bacilli  which  have  temporarily  lost  some  of 
their  faculties.  So  many  such  organisms  have  been  isolated  and  named  it  would 
be  almost  impossible  to  enumerate,  much  less  descfibe,  them  and  commonly  they 
are  referred  to  as  a  whole  as  paracolon  bacilli. 

In  addition  to  paratyphoid  A,  B,  enteritidis  of  Gaertner,  and  enteritidis 
Aertrycke,  each  of  which  is  a  distinct  species,  there  are  a  large  number  of  organ- 
isms indistinguishable  from  them  and  the  typhoid  bacillus  in  most  properties  but 
failing  to  conform  in  some  important  particular,  the  exact  status  of  these  is 
doubtful  and  as  a  whole  they  are  referred  to  as  paratyphoid  bacilli. 

For  a  more  elaborate  description  of  the  paratyphoid  bacilli  see  Archives  of  Internal  Medi- 
cine, vol.  v,  No.  3,  Mar.  15,  1910. 


CHAPTER  XXV 


DIAGNOSIS  OF  TYPHOID  FEVER  AND  FOOD-POISONING  INFECTIONS 

The  Widal  Test. — To  carry  out  this  test  one  must  have  a  24-hour-old  cul- 
ture of  the  typhoid  bacillus  free  from  clumps.  To  determine  the  presence  or 
absence  of  clumps  a  loopful  of  the  culture  is  placed  on  a  cover  glass,  the  cover  in- 
verted and  placed  on  a  hanging-drop  slide  so  that  the  fluid  is  suspended  in  the 
well.  The  preparation  is  examined  under  the  microscope  and  if  numerous, 
isolated,  actively  motile  bacilli  are  observed  the  culture  is  fit  for  use;  if  bacilli  are 
few  or  not  motile  another  culture  must  be  procured,  and  if  clumps  of  bacteria  are 
observed  they  must  be  removed  before  it  is  fit  for  agglutination  tests.  Clumps 
are  removed  by  filtration  through  sterile  filter-paper. 


*'*    * 


FIG.  23. — WIDAL  REACTION. 

Upper  half  of  field  shows  a  negative  reaction,  no  agglutination.     Lower  half  of  field  shows  a 
positive  reaction,  agglutination  of  bacilli.     (12  X  eyepiece  and  M2  oil  immersion  objective.) 

The  ear  or  finger  of  the  patient  is  cleaned  and  pricked  with  a  needle  or  lancet. 
Several  drops  to  i  cc.  of  blood  are  collected  in  a  sterile  capillary  tube,  a  Wright's 
capsule  or  upon  a  clean  glass  slide.  If  collected  in  a  tube  or  capsule  the  ends 
are  plugged  with  cotton  or  otherwise  sealed.  The  blood  coagulates  in  the  tube 
and  clear  serum  separates.  When  the  test  is  performed  the  serum  alone  is  used. 

If  collected  on  a  slide  the  blood  is  allowed  to  dry  and  when  the  test  is  per- 
formed several  drops  of  sterile  water  are  mixed  with  the  dried  blood  and  the 
liquefied  whole  blood  is  used. 

Having  obtained  the  blood  or  blood  serum  and  the  culture,  a  hanging-drop 
slide  and  several  cover  glasses  are  cleaned. 


112  MEDICAL  BACTERIOLOGY 

With  a  sterile  capillary  or  graduated  pipette  make  a  i :  20  dilution  of  the 
patient's  blood  or  serum,  using  sterile  water.  Mix  a  drop  of  the  diluted  serum 
with  an  equal  quantity  of  the  typhoid  culture  and  place  on  the  cover  glass,  invert 
it  and  place  on  the  hanging-drop  slide.  Examine  under  the  microscope  imme- 
diately and  again  at  intervals  for  J^  hour. 

If  the  bacteria  are  motile  and  not  clumped  when  first  observed  and  later 
form  clumps  agglutination  has  occurred  and  the  reaction  is  positive.  If  clump- 
ing does  not  occur  within  J^  hour  the  reaction  is  negative. 

This  test  can  be  made,  using  dead  bacilli  in  place  of  the  living  organisms. 
As  it  is  more  convenient  for  general  practitioners,  a  preparation  giving  satis- 
factory results,  known  as  "Ficker's  Typhus  Diagnosticum,"  has  been  placed  on 
the  market. 

QUANTITATIVE  AGGLUTINATION  TESTS 

Under  strict  aseptic  precautions  2  cc.  to  5  cc.  of  blood  is  withdrawn  from  a 
vein,  allowed  to  coagulate  and  the  clear  serum  obtained. 

A  dozen  perfectly  clean,  sterile,  5  cc.  capacity  test-tubes  are  placed  in  a  rack 
and  i  cc.  of  sterile  salt  solution  is  put  into  each  tube.  One-tenth  cc.  of  patient's 
serum  is  placed  in  the  first  tube  and  mixed  with  salt  solution;  then,  i  cc.  of  the 
contents  of  this  tube  is  transferred  to  tube  No.  2,  mixed  with  the  salt  solution 
and  i  cc.  is  removed  from  this  tube,  and  mixed  with  the  salt  solution  in  No.  3, 
and  so  on  until  the  last  tube  has  been  reached.  Then,  each  tube,  except  the  first 
tube,  contains  i  cc.  of  diluted  serum,  the  dilutions  from  tube  No.  2  to  tube  No. 
12  being  as  follows:  1:20,  1:40,  1:80,  1:160,  1:320,  1:640,  1:1280,  1:2560, 
1:5120,  1:10,240,  1:20,480,  1:40,960. 

One  cc.  of  a  24-hour-old  bouillon  culture  of  the  typhoid  bacillus  is  added  to 
each  tube. 

The  tubes  are  incubated  at  37°C.  for  3  hours,  and  then  examined  for  agglu- 
tination. In  this  manner  the  titre  limit  of  the  serum,  or  the  highest  dilution  in 
which  it  will  agglutinate,  is  determined. 

After  incubation,  tubes  in  which  agglutination  has  not  occurred  have  the 
same  appearance  as  before  incubation — the  fluid  is  uniformly  cloudy,  there 
is  no  precipitate.  Tubes  in  which  agglutination  has  occurred  contain  clear 
fluid  and  show  a  precipitate. 

If  the  macroscopic  appearance  is  doubtful  the  contents  of  the  tube  is 
poured  into  a  thin,  flat  dish  and  examined  with  microscope,  clumps  of 
bacteria  being  observed  if  agglutination  has  occurred. 

AGGLUTINATION  TESTS  WITH  LABORATORY  SERA 

Guinea-pigs  and  rabbits  may  be  immunized  against  the  typhoid  bacillus  by 
intraperitoneal  inoculations  with  the  typhoid  bacillus  given  as  follows: 

First. — -Injection  J^  cc.  of  24-hour-old  bouillon  culture,  heated  to  56°C.  for 
^  hour. 

Second. — 'Injection  i  cc.  of  same. 

Third. — Injection  2  cc.  of  same. 


DIAGNOSIS  OF  TYPHOID  FEVER  AND  FOOD-POISONING  INFECTIONS      113 

The  first  three  injections  are  given  at  intervals  of  48  hours. 

Several  days  after  the  third  injection  o.i  cc.  of  a  i6-hour-old  bouillon  culture, 
unheated,  is  given  and  several  days  later  0.5  cc.  of  a  i6-hour-old  bouillon  culture, 
unheated,  is  given.  Ten  days  later  the  animals  are  bled  and  the  blood  serum 
collected. 

If  the  procedure  has  been  successful,  it  will  be  discovered  by  agglutination 
tests  that  this  specific  immune  serum  in  high  dilutions  (1:20,000  to  1:50,000) 
will  agglutinate  typhoid  bacilli.  It  will  not  agglutinate  colon,  paratyphiod  A., 
paratyphoid  B,  enteritidis  of  Gaertner  nor  other  bacilli  at  all,  or  only  when 
much  less  diluted  (1:100  to  1:5000). 

.  By  immunizing  animals  in  a  like  manner  with  paratyphoid  A  bacillus  a 
specific  agglutinating  serum  for  this  organism  can  be  obtained;  by  immunizing 
with  paratyphoid  B  bacillus,  agglutinins  specific  for  it  may  be  obtained.  Like- 
wise specific  agglutinins  for  most  of  the  members  of  the  typhoid-colon  group  of 
bacteria  may  be  produced. 

When  an  organism  of  the  typhoid-colon  group  is  isolated  from  a  patient's 
blood,  urine,  feces,  or  is  found  in  water  or  food  its  nature  may  be  determined  as 
follows : 

Make  a  24-hour-old  bouillon  culture  of  the  organism;  make  dilutions  of  a 
specific  typhoid  serum  from  i :  100  to  the  titre  limit  of  the  serum.  With  the  sus- 
pected culture  and  known  serum  make  quantitative  agglutination  tests.  If  the 
organism  is  the  typhoid  bacillus  it  will  be  agglutinated  in  dilutions  up  to  the 
titre  limit  of  the  serum.  If  it  is  not  the  typhoid  bacillus  but  some  other  member 
of  the  typhoid-colon  group  it  will  not  be  agglutinated  in*  any  dilution  or  only  in 
dilutions  much  lower  than  the  dilutions  in  which  the  same  serum  agglutinated 
the  typhoid  bacillus. 

By  repeating  the  experiment  with  the  same  culture  and  a  specific  paraty- 
phoid A  serum,  and  if  necessary  again  with  paratyphoid  B  serum,  eventually  we 
will  find  a  specific  serum  which  agglutinates  the  organism  in  the  same  high  dilu- 
tion as  it  agglutinates  the  organism  with  which  the  specific  serum  was  produced 
and  the  classification  is  then  established. 

VALUE  OF  AGGLUTINATION  TESTS 

Agglutination  tests  made  with  undiluted  serum  or  diluted  less  than  1:20  are 
without  significance  for  two  reasons: 

1.  Under  such  conditions  some  sera  exert  an  antiagglutinating  effect. 

2.  The  sera  of  some  apparently  normal  non-immune  individuals  under  such 
conditions  will  agglutinate  one  or  more  of  the  members  of  the  typhoid-colon 
group. 

The  Widal  test,  made  in  dilutions  of  from  i :  20  to  i :  100,  in  suspected  typhoid 
fever  cases  gives  accurate  results  in  more  than  75  per  cent.,  some  claim  in  more 
than  90  per  cent,  of  cases;  negative  if  the  disease  is  not  typhoid;  positive  if 
the  disease  is  typhoid. 

The  sources  of  error,  other  than  bad  technique,  in  the  Widal  test,  are  as 
follows : 

8 


114  MEDICAL  BACTERIOLOGY 

During  the  first  weeks  of  typhoid  fever  the  reaction  is  often  negative;  in  rare 
cases  the  reaction  is  negative  until  the  second  or  third  week  of  the  disease  there 
are  a  few  reported  cases  in  which  the  Widal  test  has  been  negative  throughout 
the  disease;  occasionally  diseases  other  than  typhoid  fever  give  a  positive 
reaction. 

Agglutination  tests  made  with  specific  sera  and  suspected  organisms,  when 
properly  performed,  give  accurate  results  in  the  vast  majority  of  cases. 

Specific  agglutinating  sera  often  contain  group-agglutinins;  that  is,  they 
will  agglutinate  one  or  several  members  of  the  typhoid-colon  group  in  addition 
to  the  particular  organism  employed  to  produce  them.  In  practically  all  cases 
the  power  to  agglutinate  other  organisms  is  much  less  than  the  power  of  agglu- 
tinating the  particular  bacillus  which  produced  them. 

Errors  can  therefore  be  avoided  by  determining  the  highest  dilutions  in 
which  a  serum  exerts  a  group-agglutinating  action  and  those  dilutions  in 
which  its  action  is  specific. 

BLOOD  CULTURES 

In  typhoid  fever,  paratyphoid,  food  poisoning,  colon  septicemia  and  infec- 
tions with  other  members  of  the  typhoid-colon  group  of  organisms  in  which  the 
clinical  course  is  that  of  typhoid  fever,  the  offending  organism  can  be  obtained 
from  the  patient's  blood  in  all  cases  during  the  first  week  or  two  of  the  disease 
and  frequently  later.  In  severe  prolonged  cases  of  typhoid  fever  the  organism 
can  usually  be  obtained  from  the  blood  later  than  the  second  week  of  the 
disease. 

TECHNIQUE 

The  night  before  operation  the  patient's  arm  and  forearm  should  be  washed 
with  soap  and  water,  alcohol  and  1:1000  bichloride,  and  covered  from  4  inches 
below  to  4  inches  above  the  elbow  with  a  moist  bichloride  dressing  and  sterile 
bandage. 

A  flask  containing  at  least  200  cc.  of  plain  bouillon  having  a  reaction  of  -f-i-o 
or  +1.5  is  taken  to  the  bedside.  This  culture  medium  must,  of  course,  be 
sterile.  A  sterile  lo-cc.  glass  syringe  and  needle  in  a  sterile  container  is  also 
required. 

The  operator  scrubs  his  hands  with  soap  and  water  and  then  with  alcohol 
and  dries  them  on  a  sterile  towel.  The  dressings  are  removed  from  the  patient's 
arm  and  an  area  of  about  an  inch  in  diameter  over  the  most  prominent  vein 
is  painted  with  tincture  of  iodine.  A  crystal  of  carbolic  acid  is  touched  to  the 
spot  through  which  the  needle  will  be  inserted.  The  needle  is  firmly  fastened 
to  the  syringe  and  thrust  into  the  vein.  At  least  2  cc.,  better  5  cc.  of  blood  is 
drawn  into  the  syringe.  The  needle  is  withdrawn  from  the  vein,  guarded  from 
contamination  or  contact  with  anything,  and  the  contents  of  the  syringe 
emptied  into  the  culture  medium  without  any  previous  attempt  to  remove  the 
needle  from  the  syringe. 

When  the  plug  is  removed  from  the  flask  and  until  it  is  replaced,  contamina- 


DIAGNOSIS  OE  TYPHOID  FEVER  AND  FOOD-POISONING  INFECTIONS      115 

tion  of  both  the  plug  and  culture  medium  must  be  guarded  against.  As  soon  as 
the  blood  is  in  the  flask  and  the  plug  replaced,  the  flask  is  shaken  for  several 
minutes  to  thoroughly  mix  the  blood  and  culture  medium  and  to  prevent  clot- 
ting. During  the  shaking  and  transportation  care  must  be  observed  not  to 
bring  the  contents  of  the  flask  into  contact  with  the  cotton  plug. 

The  flask  is  taken  directly  from  the  bedside  to  the  laboratory  and  placed 
an  incubator  at  37°C. 

After  12  hours  the  flask  is  examined  for  bacterial  growth;  if  not  discovered 
the  flask  is  replaced  in  the  incubator  and  again  examined  every  6  hours  until 
growth  is  observed,  or  until  72  hours  have  passed.  When  the  flask  remains 
sterile  after  72  hours  it  is  strong  evidence  that  the  infection  is  not  due  to  any 
of  the  typhoid  colon  group  of  bacteria.  Evidence  of  other  infectious  disease, 
especially  tuberculosis,  should  be  sought  for. 

The  method  of  examining  the  culture  for  growth  is  as  follows: 

Shake  the  flask,  remove  the  plug,  take  out  a  drop  of  the  fluid  with  a  sterile 
platinum  loop,  replace  the  plug,  place  the  drop  of  fluid  on  a  clean  cover  glass 
and  suspend  on  a  hanging-drop  slide.  Examine  the  drop  under  the  microscope; 
if  no  organisms  are  observed  remove  and  examine  several  more  drops.  If  a 
motile  bacillus  appears  it  is  probably  the  typhoid,  paratyphoid  A,  paratyphoid 
B  or  colon  bacillus.  To  determine,  plant  a  loopful  of  the  culture  in  litmus  milk, 
make  a  stab  culture  in  litmus  lactose  agar  and  another  in  litmus  glucose  agar. 
Place  these  tubes  in  an  incubator  at  37°C.  and,  after  18  hours,  inspect  for  char- 
acteristic reactions.  * 

Instead  of  making  stab  cultures  in  litmus  lactose  agar  and  litmus  glucose 
agar,  fermentation  tubes,  containing  litmus  glucose  bouillon  and  litmus  lactose 
bouillon,  may  be  planted. 

These  three  subcultures  will  suffice,  in  the  majority  of  cases,  to  disclose  the 
nature  of  the  offending  organism.  The  reaserch  may  be  carried  further  by 
making  subcultures  in  Dunham's  solution  and  testing  for  indol,  planting  in 
litmus  bouillon  containing  saccharose,  maltose,  mannite,  levulose,  galactose, 
dulcite,  raffinose,  arabinose  and  inulin.  Agglutination  tests  with  specific  sera 
may  be  made  and  films  stained  by  Gram's  method  should  be  examined. 

In  summing  up,  it  may  be  stated  that  in  suspected  cases  of  typhoid  fever  the 
laboratory  diagnosis  should  consist  of  both  a  Widal  test  a*nd  blood  culture,  with 
the  expectation  that  one  or  the  other  be  positive  in  typhoid;  that  a  negative 
Widal,  together  with  the  occurrence  of  a  motile,  Gram  negative  organism  in  the 
culture  from  the  blood  is  suggestive  of  paratyphoid  or  colon  infection,  especially 
if  occurring  after  the  first  week  of  the  disease;  that  the  true  nature  of  the  infec- 
tion can  be  determined,  in  most  cases,  when  a  culture  is  obtained  from  the  blood, 
by  making  subcultures  in  three  different  media,  litmus  milk,  litmus  lactose  agar 
or  litmus  lactose  bouillon  and  litmus  glucose  agar  or  litmus  glucose  bouillon; 
that  repeated  failure  to  obtain  either  a  positive  Widal  or  blood  culture  in  a  sus- 
pected case  of  typhoid  fever,  indicates  infection  with  an  organism  not  belonging 
to  the  typhoid-colon  group,  usually  the  tubercle  bacillus. 


Il6  MEDICAL  BACTERIOLOGY 

EXAMINATION  OF  URINE 

Typhoid  bacilli  may  occur  in  the  urine  at  times  during  typhoid  fever,  in  the 
period  of  convalescence  and  occasionally  long  after  recovery,  and  in  carriers, 
healthy  individuals  having  no  history  of  typhoid  fever.  Indications  for  exami- 
nation of  the  urine  for  typhoid  bacilli  are  the  occurrence  of  nephritis  or  cystitis 
during  the  course  of  typhoid  fever  or  as  a  sequelae  of  the  disease,  before  the 
discharge  of  patients  recovering  from  typhoid  fever  and  those  suspected  of  being 
carriers. 

On  account  of  the  liability  of  contamination  with  colon  bacilli,  the  external 
genital  organs  should  be  thoroughly  cleansed  before  withdrawing  urine  for 
examination.  The  sample  should  be  obtained  with  a  sterile  catheter  when  the 
bladder  is  full,  and  should  pass  directly  from  the  catheter  into  a  sterile  bottle, 
and  be  examined  without  delay. 

Different  amounts  of  urine  are  planted  on  the  surface  of  separate  plates  of 
Endo's  medium — from  one  loopful  to  several  loopsful,  and  one  loopful  of  the 
sediment  obtained  by  centrifugalizing  15  cc.  of  urine.  The  plants  are  made  by 
drawing  the  loop  over  the  medium  in  the  form  of  the  letter  Z. 

The  plates  are  incubated  at  37°C.  for  24  hours  and  then  examined  for  isolated 
colonies. 

Sterile  Endo's  medium  is  colorless  or  a  very  faint  pink  Colonies  of  typhoid 
and  paratyphoid  bacilli  do  not  change  the  color  of  the  medium  and  themselves 
appear  colorless  or  whitish.  Colon  colonies  cause  the  medium  surrounding 
them  to  turn  red  a*hd  the  colonies  may  appear  colorless  or  pinkish.  If  typhoid- 
like  colonies  appear  on  Endo's  medium,  they  are  removed  with  a  sterile  loop 
and  planted  in  Dunham's  solution,  litmus  milk,  litmus  glucose  and  litmus 
lactose  media.  The  subcultures  are  incubated  at  37°C.  for  24  hours  and  then 
examined  for  characteristic  changes. 

Colon  bacillus  infections  of  the  bladder  or  kidneys  are  common.  In  suspected 
cases  the  urine  is  examined  by  planting  on  Endo's  agar  and  making  subcultures 
as  just  described. 

EXAMINATION  OF  FECES 

Feces  always  contain  large  numbers  of  colon  bacilli,  which  makes  isolation 
of  other  organisms  difficult. 

Typhoid  bacilli  occur  in  the  feces  of  typhoid  fever  patients,  some  convales- 
cents, and  occasionally  for  months  or  years  after  recovery  from  typhoid  fever. 
Typhoid  bacilli  also  occur  in  the  feces  of  carriers. 

Feces  should  be  examined  for  typhoid  bacilli  before  the  discharge  of  conva- 
lescent typhoid  patients  and  when  a  person  is  suspected  of  being  a  carrier. 

The  best  time  to  examine  feces  for  typhoid  bacilli  is  immediately  after  they 
are  expelled  from  the  rectum;  attempts  made  several  hours  later  are  usually 
futile.  On  account  of  the  great  preponderance  of  colon  bacilli  in  feces  it  is 
necessary  to  first  plant  the  specimen  in  a  medium  that  will  favor  the  growth  of 
typhoid  and  paratyphoid  bacilli  and  retard  the"  growth  of  the  colon  bacillus. 
From  this  primary  culture  in  enriching  medium  subcultures  are  made. 


FIG.  24. — PLATE  CULTURE,  ENDO'S  MEDIUM  (AGAR)  SHOWING  TYPHOID  AND  COLON 

COLONIES. 

The  colon  colonies  appear  red  surrounded  by  a  red  zone  in  the  culture  medium.     The 
typhoid  are  the  transparent  colonies. 


DIAGNOSIS  OF  TYPHOID  FEVER  AND  FOOD-POISONING  INFECTIONS      II 7 

Some  observers  claim  the  addition  of  caffeine  (6  cc.  of  sat.  aqueous  sol.  to 
100  cc.  of  medium)  inhibits  colon  growth  without  affecting  the  typhoid. 
Crystal  violet,  Cong,  red  and  brilliant  green  exert  a  similar  effect. 
Teague  and  Clurman  recommend  the  following  enriching  medium: 

Meat  infusion  bouillon,  1000  cc. 

Gelatin,  100  Gm. 

Cong,  red  (2  per  cent,  aqueous  sol.),  4  cc. 

Brilliant  green  0.083  Per  cent. 
Tube,  10  cc.  to  each  tube,  and  sterilize. 

Just  before  inoculation  add  i  cc.  of  the  following  to  each  tube: 

Bromoform,  20  cc. 

Sterile  distilled  water,  100  cc. 

Shake  and  allow  to  stand  over  night.     Avoid  globules  of  bromoform  on  sur- 
face and  on  bottom  when  removing  fluid  for  addition  to  media. 

Hall  in  the  Berliner  klin.  Wochenschrift,  Dec.  27,  1915,  reports  excellent 
results  in  the  isolation  of  typhoid  bacilli  from  feces  by  mixing  them  with  benzin 
or  with  petroleum-ether  and  then  plating.  He  states  that  this  will  kill  all  colon 
bacilli  without  injuring  the  typhoid  or  interfering  with  their  propagation. 


TECHNIQUE 

The  evening  before  feces  are  collected  for  examination  the  patient  is  given  a 
dose  of  salts. 

The  patient  defecates  in  a  clean  bed  pan  or  sterile  towek. 

Liquefy  several  tubes  of  Teague  and  Clurman's  medium,  add  o.i  cc.  of 
emulsified  feces  to  one  tube,  shake  tube  to  evenly  distribute  the  feces,  transfer 
o.i  cc.  of  the  contents  of  this  tube  to  a  second  tube,  shake  second  tube  to  evenly 
distribute  bacteria  and  transfer  o.i  cc.  of  its  contents  to  third  tube  which  is  also 
shaken. 

Incubate  at  37°C.  until  growth  is  observed,  then  at  once  plate. 

Pour  Conradi  and  Drigalski's  or  Endo's  medium  into  a  number  of  petri 
dishes  and  when  solidified  plant  several  plates  from  each  tube  by  removing  a 
loopful  of  fluid  from  a  tube  and  drawing  it  over  the  surface  of  one  plate  after 
another  without  recharging  the  loop.  The  loop  should  be  drawn  over  the  sur- 
face of  the  agar  in  the  form  of  the  letter  Z. 

Incubate  at  37°C.  until  growth  appears.  Examine  plates  showing  discrete 
colonies  and  any  having  the  typical  appearance  of  typhoid  colonies  are  removed 
and  planted  in  plain  bouillon;  after  incubation  transplants  from  the  bouillon 
are  made  into  differentiating  media — trypsinized  peptone  water  (for  indol), 
litmus  glucose  broth,  litmus  lactose  broth  and  litmus  milk. 

The  method  of  examining  urine  and  feces  for  other  members  of  the  typhoid 
colon  group  is  exactly  the  same  as  when  examining  for  the  typhoid  bacillus. 


nS 


MEDICAL  BACTERIOLOGY 


EXAMINATION  OF  PUS,  SPUTUM  AND  SEROUS  EFFUSIONS 

When  abscess  formation,  peritonitis,  pleurisy,  meningitis  or  pneumonia 
occurs  during  the  course  of  typhoid  fever  or  under  circumstances  which  suggest 
the  typhoid  bacillus  as  a  probable  cause,  some  of  the  pus  or  fluid  exudate  should 
be  planted  on  the  surface  of  Endo's  agar  plates  with  a  platinum  loop,  incu- 
bated, and  if  typhoid-like  colonies  appear  they  are  subcultured  and  examined 
microscopically. 

EXAMINATION  OF  BILE 

A.  L.  Garbat  states  that  the  urine  may  contain  bacilli  when  the  feces  and 
bile  do  not;  that  the  feces  may  contain  them  when  the  urine  and  bile  do  not; 
that  the  bile  alone  may  reveal  bacteria;  that  all  three— urine,  feces  and  bile — 
should  be  examined  before  typhoid  patients  are  discharged  and  when  an 
individual  is  suspected  of  being  a  carrier. 

He  states  that  the  examination  of  bile  for  typhoid  bacilli  is  easier  than  the 
examination  of  feces  and  frequently  yields  pure  cultures  of  them  when  examina- 
tion of  feces  discloses  none.  He  recommends  the  following  technique : 

Have  patient  swallow  an  Einhorn  duodenal  tube  when  retiring  at  night;  give 
a  liquid  breakfast  the  following  morning;  i  J^  hours  after  breakfast  pour  10  ounces 
of  sterile  normal  salt  solution  through  tube;  J/2  h°iir  later  withdraw  bile  from 
tube  with  a  sterile  syringe  and  plant  the  contents  of  syringe  in  plain  broth  and 
on  agar;  if  growth  occurs,  make  subcultures  to  determine  whether  typhoid  or 
other  bacilli  have  been  obtained. 


Bacillus 

Size 

Motility 

Gram's 
method  of 
staining 

Litmus  milk 

Colon  

2to3/iXsto8/x 
2t03/*X5to8/i 
2to3/iXsto8M 
2t03/*X5to8/i 

2to3A*X5to8/i 

Motile 
Motile 
Motile 
Motile 

Motite 

Negative 
Negative 
Negative 
Negative 

Negative 

Acidulated  and  coagulated 
Not  acidulated  and  not  coagulated 
Acidulated,  not  coagulated 
Acidulated,    later    turns   blue,    not 
coagulated 
Acidulated,   later   turns   blue,    not 
coagulated 

Tvohoid 

Paratyphoid  A 
Paratyphoid  B 

Bacillus 

Glucose 

Lactose 

Indol   . 

Agglutination 

Colon    

Acid  and  gas 

Acid,   no  gas 
Acid  and  gas 
Acid,  and  gas 
Acid  and  gas 

Acid  and  gas 

No  acid,  no  gas 
No  acid,  no  gas 
No  acid,  no  gas 
No  acid,  no  gas 

Produces  indol 

No  indol 
No  indol 
No  indol 
No  indol 

Is  not  agglutinated  with 
specific  typhoid  or  para- 
typhoid serum 
Agglutinated  with  specific 
typhoid  serum 
Agglutinated  with  specific 
paratyphoid  A  serum 
Agglutinated  with  specific 
paratyphoid  B  serum 
Agglutinated  with  specific 
Gaertner  serum 

Typhoid 

Paratyphoid  A 
Paratyphoid  B 
Gaertner  

CHAPTER  XXVI 


DYSENTERY  BACILLI 

Dysentery  is  a  disease  produced  by  ameba  and  by  a  number  of  closely 
related  bacilli.  The  bacillary  form  of  the  disease  occurs  sporadically  and 
in  epidemics  in  many  parts  of  the  temperate  and  torrid  zones. 

The  distribution  of  the  various  types  of  dysentery  bacilli  is  not  fully 
known,  and  it  may  be  the  same  for  each. 

Morphology. — The  dysentery  bacilli  have  the  same  size  and  shape  as  the 
typhoid  bacillus;  most  of  them  are  not  motile. 

In  staining,  growth  in  bouillon,  agar,  gelatin,  blood  serum  and  potato 
they  are  indistinguishable  from  the  typhoid  bacillus;  they  differ  from  is 
and  from  each  other  in  affect  on  carbohydrates  and  agglutination  reaction. 

HISS  AND  ZINSSER'S  CLASSIFICATION  * 


Bacillus 


Dextrose,  levulose,  galactose 


Dysentery  (Shiga-Kruse  Type) 
Dysentery  (Hiss-Russel  Type) 
Dysentery  (Flexner  Type) 
Dysentery  ("Rosen") 


Acid  formed  but  no  gas 
Acid  formed  but  no  gas 
Acid  formed  but  no  gas 
Acid  formed  but  no  gas 


Mannite 

Maltose 

Lactose 

Saccharose                       Dextrin 

I 

No  acid,  no  gas 
Acid,  no  gas 
Acid,  no  gas 
Acid,  no  gas 

No  acid,  no  gas 
No  acid,  no  gas 
Acid,  no  gas 
Acid,  no  gas 

No  acid,  no  gas 
No  acid,  no  gas 
No  acid,  no  gas 
Acid,  no  gas 

No  acid,  no  gas 
No  acid,  no  gas 
Acid,  no  gas 
No  acid,  no  gas 

No  acid,  no  gas 
No  acid,  no  gas 
Acid,  no  gas 
Acid,  no  gas 

Litmus  milk 

Primary  acidity  followed  by  alkalinity 
Primary  acidity  followed  by  alkalinity 
Primary  acidity  followed  by  alkalinity 

Acid 

Non-motile 
Non-motile 
Non-motile 

Slightly  motile 

Maltose  fermented  after  some  days 

With  few  exceptions  dysentery  bacilli  do  not  form  indol.  They  do  not 
form  spores. 

Resistance. — Outside  the  human  body  the  resistance  of  the  dysentery 
bacilli  to  germicidal  agents  is  much  less  than  that  of  the  typhoid  bacillus. 

*"A  Text  Book  of  Bacteriology"  Hiss  and  Zinsser. 

119 


120  MEDICAL  BACTERIOLOGY 

They  cannot  be  found  in  feces  48  hours  after  defecation,  due  to  the  an- 
tagonistic action  of  the  colon  bacillus.  In  water  saprophytic  bacteria  cause 
them  to  disappear  in  a  few  days.  They  dry  out  in  cultures  after  3  or  4  weeks, 
unless  transplanted.  In  a  moist  state  55°C.  kills  them  in  less  than  an  hour. 
In  a  hot-air  sterilizer  they  are  destroyed  in  less  than  an  hour  at  ioo°C. 

Toxin. — Dysentery  bacilli  form  an  intracellular  toxin. 

Agglutinins,  precipitins  and  amboceptors  occur  in  the  serum  of  dysentery 
patients  and  immunized  animals.  The  agglutinins  are  specific;  the  serum  from 
a  patient  agglutinates  the  type  of  bacillus  causing  his  condition,  but  none  of 
the  other  dysentery  bacilli.  The  amboceptors  give  a  group  reaction;  they  will 
fix  complement  when  any  type  of  dysentery  bacillus  is  used  as  antigen. 

Pathogenesis — Bacillary  dysentery  results  from  ingestion  of  polluted  water 
or  food.  The  bacteria  are  usually  confined  in  the  primary  seat  of  infection — 
the  intestines — it  is  possible  that  in  some  cases  they  may  migrate  to  the  gall- 
bladder, but  there  is  not  a  bacteremia  as  in  typhoid  fever.  They  escape  in  the 
feces  and  bloody  mucous  flux  of  the  disease.  Bacteria  are  most  readily  found 
in  the  stools  during  the  first  days  of  the  disease  and  disappear  after  several 
weeks. 

Different  types  of  dysentery  bacilli  show  different  degrees  of  virulence,  and 
it  is  said  that  the  Shiga  type  is  the  most  virulent. 

Relatively  large  amounts  of  living  bouillon  cultures  introduced  through  the 
esophagus  produce  signs  and  lesions  of  the  disease  in  guinea-pigs  and  rabbits 
similar  to  the  manifestations  observed  in  man. 

Diagnosis. — The  bacteriological  diagnosis  of  dysentery  consists  in  the 
examination  of  feces  microscopically  and  by  culture  and  examination  of  blood 
serum  for  agglutinins. 

Feces. — Have  the  patient  defecate  into  a  sterile  bed  pan,  immediately  pick 
out  a  mucous  shred  and  place  it  on  a  slide  and  let  a  cover  glass  fall  upon  it; 
then,  examine  under  the  microscope  for  ameba.  When  ameba  are  present 
they  may  not  be  found  until  a  number  of  slides  have  been  examined.  If  several 
slides  made  from  mucus  fail  to  show  them,  slides  should  be  made  from  the  fluid 
portion  of  the  feces  and  from  solid  particles  by  dipping  a  camel's-hair  brush 
into  it  and  gently  smearing  the  slide. 

After  making  a  microscopic  examination  for  ameba,  or  before  doing  so, 
a  shred  of  the  gelatinous-like  substance  or  blood  matter  found  in  such  stools  is 
sought  for,  removed  with  a  platinum  loop,  washed  with  sterile  water  and  then 
drawn  across  the  surface  of  a  number  of  plates  of  Endo's  agar.  The  plates  are 
incubated  at  37°C.  for  1 8  to  24  hours.  Red  colonies  are  passed  by;  any  color- 
less colonies  that  appear  are  removed  and  transplanted  into  litmus  milk, 
Dunham's  solution,  and  litmus  bouillon,  containing  various  carbohydrates, 
used  to  differentiate  dysentery  bacilli.  The  subcultures  are  incubated  at 
37°C.  and  inspected  each  day  for  several  days.  If  a  dysentery  bacillus  is  found, 
a  24-hour-old  bouillon  culture  is  used  to  make  agglutination  tests  with  the 
patient's  serum. 

Agglutination  Tests. — The  patient's  finger  is  asepticized,  pricked  with  a 


DYSENTERY  BACILLI  121 

sterile  needle  and  several  drops  or  more  of  blood  obtained  in  a  sterile  capillary 
tube.  A  i :  10  dilution  of  the  serum  is  made  with  normal  salt  solution. 

One  drop  of  this  i :  10  dilution  of  serum  is  mixed  with  i  drop  of  a  24-hour- 
old  bouillon  culture  of  a  known  dysentery  bacillus  and  placed  on  a  hanging- 
drop  slide  and  examined  for  agglutination,  as  in  making  a  Widal  test. 

If  a  negative  result  is  obtained  the  test  should  be  repeated  with  as  many 
different  types  of  dysentery  bacilli  as  one  may  have  at  hand. 

A  positive  reaction  establishes  the  diagnosis.  Negative  reactions  do  not 
indicate  the  absence  of  bacillary  dysentery  unless  made  with  all  the  different 
types  of  dysentery  bacilli.  It  is  to  be  remembered  that  agglutinins  are  not 
present  in  the  blood  during  the  first  few  days  of  the  disease  and  that  bacteria 
cannot  be  isolated  from  the  stools  after  the  third  week  of  the  disease. 


CHAPTER  XXVII 

SPIRILLUM   CHOLERA  ASIATICS,  VIBRIO  CHOLERA 
ASIATICS  (COMMA  BACILLUS) 

The  cholera  spirillum  occurs  in  water  polluted  with  dejecta  from  cholera 
patients  and  from  this  source  probably  gets  to  some  articles  of  food.  Epidemics 
of  cholera  occur  from  time  to  time  in  all  densely  populated  regions  where 
pollution  of  drinking  water  occurs,  especially  in  the  temperate  zones. 

Morphology. — The  cholera  spirillum  usually  appears  as  a  curved  rod,  from 
1.5  to  3  p  long,  0.5  fj,  wide;  it  is  to  this  common  appearance  that  the  descriptive 
name  comma  bacillus  is  due.  Long  spirals  are  also  observed,  having  two,  three 
or  more  curls  like  a  corkscrew.  The  cholera  spirillum  has  a  single  terminal 
flagellum  and  is  actively  motile. 

Staining. — The  cholera  spirillum  stains  with  all  the  usual  stains  and  is 
Gram  negative. 

Growth. — The  cholera  spirillum  is  aerobic  and  to  a  slight  degree  facultative 
anaerobic.  It  grows  at  temperatures  between  i5°C.  and  4o°C.,  best  at  37°C. 

Bouillon  incubated  at  37°C.  shows  cloudiness  in  6  to  8  hours;  later,  a  deli- 
cate whitish  pellicle  forms.  In  48  to  72  hours  a  flocculent  precipitate  occurs. 

Agar. — Round,  grayish  colonies,  with  granular  centers  and  smooth  edges, 
appear  in  24  hours.  Growth  on  agar  is  luxuriant  and  may  cover  the  entire 
surface  with  a  whitish  film  in  1 8  to  36  hours. 

Gelatin.— In  gelatin  stabs  growth  first  appears  at  the  surface  of  the  stab, 
causing  a  zone  of  liquefaction;  growth  progresses  along  the  stab,  more  slowly 
the  further  from  the  surface,  causing  a  funnel-shaped  tract  of  liquefaction. 
Eventually  all  the  gelatin  is  liquefied. 

On  gelatin  plates  yellowish,  granular,  irregular  colonies,  surrounded  with  a 
more  highly  refractile,  whitish  zone  appear  in  24  hours. 

The  medium  is  liquefied. 

Potato  shows  an  abundant,  clear,  brownish  growth  if  the  medium  is  alkaline. 
On  neutral  or  acid  potato,  growth  is  scant  or  absent. 

Milk  is  coagulated  by  some  and  not  coagulated  by  other  strains  of  the 
cholera  spirillum. 

Indol  is  nearly  always  produced. 

There  is  no  spore  formation. 

Fermentation  of  all  the  sugars  in  common  use  occurs. 

Dunham's  solution  is  especially  favorable  for  the  cultivation  and  isolation 
of  the  cholera  spirillum.  Incubated  at  37°C.,  growth  accumulates  at  the 
surface  in  6  to  12  hours. 

Resistance. — The  cholera  spirillum  is  less  resistant  to  germicidal  agents 
than  the  typhoid  bacillus.  Freezing  does  not  injure  it.  Drying  rapidly  de- 

122 


SPIRILLUM    CHOLERA   ASIATICS  123 

stroys  it.  In  a  moist  state  exposure  to  55°C.  kills  in  J^  hour.  In  feces  and  in 
water  containing  saprophytic  bacteria  they  are  destroyed  in  a  few  days. 

Cholera  vibrio  survives  in  bay  water  7  to  49  days  but  will  not  multiply  in  it. 

Toxin. — The  cholera  bacillus  produces  an  intracellular  toxin  or  toxins. 

The  injection  of  living  or  dead  cholera  organisms  into  the  subcutaneous 
tissues,  peritoneal  cavity  or  into  a  vein  produces  immunity.  The  serum  of 
immunized  animals  contains  agglutinins  and  amboceptors  for  the  cholera 
spirillum. 

Agglutinins  also  occur  in  the  serum  of  many  cholera  patients. 

Pathogenesis. — Different  strains  of  the  cholera  spirillum  show  different 
degrees  of  virulence.  The  cholera  spirillum  enters  the  body  through  the  ali- 
mentary canal  and  lodges  in  the  intestine;  other  tissues  are  not  invaded,  but 
the  toxin  is  absorbed  and  passes  into  the  general  circulation.  The  infecting 
organisms  are  discharged  in  the  feces  where  they  may  be  found  in  abundance. 
The  spirillum  may  also  be  found  in  the  rectum  and  feces  of  carriers. 

While  cholera  is  a  disease  of  man  exclusively,  it  can  be  simulated  by  artificial 
means  in  animals. 

Virulent  cultures  of  the  cholera  spirillum  injected  into  the  peritoneal  cavity 
of  a  guinea-pig  usually  cause  peritonitis,  toxemia  and  death. 

Diagnosis.— There  are  so  many  vibriones  found  in  water,  and  occasionally 
in  feces,  which  are  indistinguishable  from  the  cholera  spirillum  in  morphology, 
staining,  growth  on  culture  media  and  results  from  animal  inoculation  tests,  that 
identification  of  the  cholera  spirillum  necessitates  agglutination  tests  and  is 
often  difficult. 

Isolation  of  the  cholera  spirillum  from  other  organisms,  occurring  with  it  in 
water  and  feces,  is  favored  by  the  luxuriant  and  more  r  apid  growth  of  the 
cholera  spirillum  in  certain  liquid  and  solid  media,  notably  Dunham's  solution. 
A  valuable  review  of  these  selective  media  may  be  found  in  Hygienic  Laboratory 
Bulletin,  No.  91,  1913,  U.  S.  Public  Health  Service. 

The  stools  of  cholera  patients  contain  many  cholera  organisms  and  should  be 
examined  immediately  after  passage  as  follows: 

Technique. — Take  a  loopful  of  the  dejecta  and  place  in  a  tube  containing 
10  cc.  of  Dunham's  solution.  Incubate  the  tube  at  37°C.  for  6  hours.  Remove 
a  loopful  of  the  culture  from  the  top  of  the  medium  and  draw  the  loop  across 
the  surface  of  several  plates  without  recharging  it.  Plain  agar,  plain  gelatin 
or  Dieudonne's  alkaline  blood  may  be  used  for  plating.  Incubate  agar  plates 
at  37°C.  and  gelatin  plates  at  22°C.  to  25°C.  After  16  hours  examine  plates 
and  remove  any  colonies  that  appear  to  be  cholera  spirillum;  transplant  into 
Dunham's  solution,  incubate  at  37°C.  for  6  or  8  hours,  remove  a  loopful  and 
make  a  hanging  drop  preparation.  Examine  with  the  microscope  and  if  a 
motile  spirillum  is  found  in  pure  culture,  make  transplants  into  milk  on  plain 
agar  and  into  Dunham's  solution.  Incubate  for  24  hours  at  37°C.  Test  the 
Dunham's  solution  for  indol  and  the  agar  growth  for  Pfeiffer's  reaction  as  fol- 
lows: Scrape  half  the  24-hour-old  growth  off  th'e  agar  and  emulsify  in  2  cc. 
of  sterile  bouillon,  make  a  hanging-drop  and  see  that  the  emulsion  is  free  from 


124  MEDICAL  BACTERIOLOGY 

clumps  and  that  the  organisms  are  motile.  Inject  the  2  cc.  of  emulsion  into 
the  peritoneal  cavity  of  a  guinea-pig  that  has  been  immunized  against  the 
cholera  spirillum.  Ten  minutes  later  withdraw  several  drops  of  the  peritoneal 
fluid,  place  on  hanging-drop  slide  and  examine  with  the  microscope.  If  the 
organism  injected  into  the  pig  was  the  cholera  spirillum  it  will  have  lost  its 
motility  and  will  appear  as  granular  dots.  If  it  was  not  the  cholera  spirillum 
it  will  appear  unaltered,  morphology  and  motility  the  same  as  before  injection. 
The  isolation  of  the  cholera  spirillum  from  water  will  be  described  in  the 
chapter  devoted  to  the  examination  of  water. 


CHAPTER  XXVIII 
MICROCOCCUS  MELITENSIS 

Micrococcus  melitensis,  first  found  in  Malta,  has  been  reported  as  occurring 
in  many  regions  adjacent  to  the  Mediterranean,  also  in  China,  Central  Africa 
and  England,  and  may  have  a  still  wider  distribution. 

This  organism  is  found  in  the  soil  of  regions  where  the  disease  prevails  and 
in  the  milk  of  infected  goats  and  the  urine  of  infected  patients. 

Morphology. — The  micrococcus  melitensis  is  a  small,  round  or  oval  organ- 
ism arranged  singly,  in  pairs  and  short  chains,  the  latter  often  parallel  to  each 
other.  According  to  early  observers  this  organism  is  a  coccus.  Recent  authori- 
ties classify  it  as  a  bacillus.  It  is  a  non-motile,  stains  with  the  usual  anilin 
stain  and  is  Gram  negative. 

Growth. — Micrococcus  Melitensis  is  an  obligate  aerobe  and  grows  best  at 
37°C. 

Bouillon. — Incubated  at  37°C.  s'hows  cloudiness  in  3  to  5  days. 

Agar. — Small  white  colonies  appear  in  3  to  5  days.  Growth  is  most  abundant 
on  glycerin  agar,  even  on  this  medium  it  is  scant. 

Gelatin. — No  apparent  growth  occurs  on  this  medium,  it  is  not  liquefied. 

Potato. — Does  not  show  growth. 

Milk  is  not  acidulated  and  is  not  coagulated. 

Indol  is  not  formed,  spores  are  not  formed,  sugars  are  not  fermented. 

Resistance. — Micrococcus  melitensis  is  somewhat  more  resistant  to  the 
various  germicidal  agencies  than  the  typhoid  bacillus. 

Toxin. — Toxin  production  in  culture  is  slight. 

Agglutinins. — Specific  agglutinins  occur  in  the  blood  of  immunized  animals. 

The  saliva,  blood,  and  sometimes  the  urine,  of  infected  persons  will  aggluti- 
nate the  micrococcus  melitensis  in  low  dilutions  after  the  first  week  of  the 
disease. 

Pathogenesis. — The  micrococcus  melitensis  produces  a  disease  in  man 
known  as  Malta  fever  or  Mediterranean  fever,  a  condition  simulating  typhoid 
fever  or  malarial  fever.  The  majority  of  cases  result  from  drinking  the  milk 
of  goats  infected  with  the  micrococcus  melitensis;  some  result  from  contact  with 
patients,  especially  when  handling  urine,  as  in  nursing.  It  is  possible  that 
infection  may  be  acquired  from  other  sources,  as  dust  and  soil. 

Diagnosis. — Diagnosis  is  based  on  the  discovery  of  the  organism  in  the  urine 
and  its  agglutination  with  the  serum  of  an  immunized  animal. 

After  the  first  week  of  the  disease  the  patient's  serum  may  be  tested,  in 
dilutions  of  1:10,  1:20  and  1:40  for  agglutinations. 

125 


CHAPTER  XXIX 
BACILLUS  PYOCYANEUS' 

The  bacillus  pyocyaneus  is  primarily  a  saprophytic  organism  and  occurs  in 
dust,  soil  and  water  and  substances  contaminated  by  them. 

Morphology. — Bacillus  pyocyaneus  is  i  to  2  n  long  and  0.5  /z  wide,  sometimes 
as  in  old  broth  cultures  elongated  forms  are  observed.  It  is  arranged  singly  and 
in  short  filaments  and  is  actively  motile. 

Staining. — It  stains  with  all  the  usual  anilin  stains  and  is  Gram  negative; 
old  cultures  (involution  forms)  may  show  granular  or  irregular  staining. 

Growth. — Bacillus  pyocyaneus  is  an  aerobic  and  facultative  anaerobic  organ- 
ism growing  at  temperatures  between  i8°C.  and  43°C.,  best  at  37°C.  Culti- 
vated under  aerobic  conditions,  it  produces  pigment-pyocyanine.  This  pig- 
ment diffuses  through  the  medium  containing  the  bacilli  and  gives  it  a  greenish 
or  bluish  tint;  This  pigment  can  be  separated  into  two  or  three  portions  as 
follows: 

To  a  bouillon  culture  showing  color  add  about  one-tenth  its  volume  of 
chloroform,  shake,  allow  to  stand  at  rest  until  chloroform  precipitates.  The 
chloroform  is  a  deep  blue  and  the  upper  part  of  the  bouillon  shows  a  fluorescent 
green. 

Bouillon. — Incubated  at  37°C.  becomes  cloudy  in  12  hours;  then  pigment 
begins  to  appear,  at  first  at  the  surface,  later  throughout  the  medium.  After 
several  days  a  thick,  whitish  pellicle  forms  on  the  surface;  later  an  abundant 
precipitate  is  thrown  down  and  the  medium  changes  from  greenish-blue  to 
yellowish-brown. 

Agar. — Growth  appears  in  24  hours  and  covers  the  entire  surface  in  several 
days  and  the  agar  attains  a  greenish  color. 

Gelatin  surface  cultures  after  48  hours  at  2o°C.  to  25°C.,  show  small,  round 
yellowish  granular  colonies;  later  the  dense  central  portion  of  the  colonies  is 
surrounded  by  a  thin,  granular  filamentous  zone.  Liquefaction  begins  around 
the  colonies  and  rapidly  involves  the  entire  medium.  The  gelatin  is  tinted  like 
agar. 

Gelatin. — Stab  cultures  show  growth  along  the  stab  in  48  hours.  The  gelatin 
is  tinted  green  and  liquefied. 

Potato. — A  thick  brown  surface  growth  develops  in  2  or  3  days. 

MacConkey's  bile-salt  medium  shows  gas  formation  and  a  decided  greenish 
tint  in  24  to  48  hours. 

Bacillus  pyocyaneus  does  not  form  spores. 

Toxin. — Bacillus  pyocyaneus  forms  an  intracellular  toxin  capable  of  produc- 
ing effect  similar  to  that  caused  by  the  living  bacillus. 

Agglutinins  are  present  in  the  blood  of  immunized  animals. 

126 


BACILLUS   PYOCYANEUS  127 

Resistance. — Bacillus  pyocyaneus  is  somewhat  more  resistant  to  heat  and 
chemical  germicides  than  the  typhoid  bacillus.  Soil  seems  to  be  its  normal 
abode  and  it  survives  in  water  longer  than  either  the  typhoid  or  colon  bacillus. 

Pathogenesis. — Bacillus  pyocyaneus,  present  in  eggs,  milk,  ice  cream  and 
other  articles  of  food,  for  a  long  time  before  consumption,  can  produce  changes 
which  result  in  acute  gastroenteritis  or  toxemia  when  such  food  is  eaten. 

It  may  produce  gastro-enteritis  especially  in  debilitated  children. 

Bacillus  pyocyaneus  is  frequently  present  upon  the  skin  and  occasionally  in 
the  mouth  or  nose  of  healthy  people  without  causing  ill  effect.  Healthy  indi- 
viduals seem  to  be  immune  to  this  organism.  But  when  injury  or  infection 
with  some  other  organism  reduces  the  vitality  to  a  marked  degree,  then  bacillus 
pyocyaneus  may  enter  the  devitalized  part  and  further  aggravate  the  condition. 
Thus,  it  is  most  commonly  found  in  chronic  suppurative  conditions,  in  advanced 
phthisis  and  extensive,  grossly  infected  wounds. 

Diagnosis. — Pus  containing  bacillus  pyocyaneus  usually  has  a  distinct  green- 
ish color  which  suggests  its  presence.  If  the  pus  is  smeared  on  a  slide,  fixed  by 
heat  and  stained  by  Gram's  method  and  a  short,  slim,  Gram  negative  bacillus 
found  it  is  sufficient  to  indicate  the  presence  of  bacillus  pyocyaneus. 

More  exact  identification  may  be  obtained  by  inoculating  bouillon,  agar  or 
gelatin  and  observing  the  pigment  formation.  It  should  be  remembered  in  this 
connection  that  cultures  direct  from  tissue  or  pus  sometimes  do  not  show  pig- 
ment production  until  subcultured  once  or  twice;  that  pigment  appears  only 
under  aerobic  conditions. 


CHAPTER  XXX 
BACILLUS  PROTEUS  VULGARIS 

Bacillus  proteus  vulgaris  is  one  of  the  common  organisms  of  putrefaction. 
It  occurs  in  air,  soil  and  water,  and  is  present  wherever  putrefaction  takes  place. 
It  splits  proteids  into  their  simplest  components. 

In  morphology,  motility  and  staining  it  is  indistinguishable  from  the  colon 
bacillus.  It  is  differentiated  from  the  colon  bacillus  by  the  following 
characteristics: 

Optimum  growth  at  25°C. 

Liquefies  gelatin  and  blood  serum. 

In  milk  it  causes  coagulation  and  then  liquefies  the  clot. 

It  is  agglutinated  with  the  serum  of  immunized  animals. 

Toxin. — Bacillus  proteus  vulgaris  produces  little,  if  any,  toxin. 

Pathogenesis. — This  organism  is  not  pathogenic  for  man,  but  is  of  impor- 
tance on  account  of  the  frequency  with  which  it  contaminates  andputrefiesjneat 
and  other  putrescible  foods  exposed  to  air  and  stored  at  room  temperature.  In 
the  process  of  putrefaction  it  liberates  compounds,  some  of  which  are  toxic. 


128 


CHAPTER  XXXI 
BACILLUS  LACTIS  AEROGENES  AND  BACILLUS  BULGARICUS 

Bacillus  lactis  aerogenes  is  widely  distributed  in  air,  water  and  soil.  It  is 
almost  constantly  present  in  milk  and  the  intestinal  canal  of  man.  This  organ- 
ism is  the  chief  cause  of  souring  of  milk.  It  produces  large  quantities  of  lactic 
acid  and  can  survive  in  media  which  it  has  made  strongly  acid. 

Escherich  described  this  organism  as  a  distinct  entity  and  some  still  consider 
it  such.  At  the  present  time  many  hold  the  view  that  bacillus  lactis  aerogenes 
and  bacillus  Friedlander  are  two  different  organisms  belonging  to  a  numerous 
group  the  members  of  which  are  closely  related  in  morphology,  growth  on  culture 
media,  etc. 

"No  valid  distinction  can  now  be  drawn  between  the  pneumobacillus  and 
the  bacillus  described  by  Escherich  as  the  bacillus  lactis  aerogenes;  the  proof 
of  their  identity  was  sketched  by  Denys  and  Martin  and  extended  by  Grinbert 
and  Legros.  These  researches  were  confirmed  by  Bertarelli;  he  considered  the 
bacillus  lactis  aerogenes  to  be  merely  a  variety  of  pneumobacillus"  (Besson). 

BACILLUS  BULGARICUS 

Bacillus  Bulgaricus,  originally  isolated  from  and  identified  as  the  dominat- 
ing organism  in  sour  milk,  in  Bulgaria,  is  a  harmless,  non-pathogenic  organism 
said  to  differ  from  other  tsaprophy tes  found  in  milk  in  the  following  particulars : 

First,  it  does  not  putrefy  milk;  second,  it  produces,  in  milk,  a  greater  amount 
of  lactic  acid,  in  a  shorter  time,  than  other  bacteria;  third,  it  does  not  produce 
changes  in  milk  that  injure  its  food  value. 

When  ingested  alive,  the  bacillus  Bulgaricus  is  supposed  to  survive  a  suffi- 
cient time  to  produce  enough  lactic  acid  to  destroy  putrefactive  bacteria  and 
thus  preclude  or  arrest  deleterious  putrefaction  in  the  intestinal  canal  of  man. 

Morphology. — Four  to  8  ju  long,  0.5  to  i.o  n  wide,  arranged  singly,  in  pairs, 
end  to  end  and  in  filaments;  sluggish  motility. 

Staining. — Stains  with  the  usual  anilin  dyes.  Young  cultures  are  Gram 
positive;  older  cultures  show  both  Gram  positive  and  negative  organisms  and 
some  which  show  granular  staining  with  methylene  blue. 

Growth. — Bacillus  Bulgaricus  is  an  aerobic  and  facultative  anaerobic 
organism  growing  best  at  42°C.  It  grows  poorly  or  not  at  all  in  plain  bouillon, 
agar  and  gelatin. 

In  milk,  whey,  whey-bouillon  and  whey-agar,  abundant  growth  occurs  in 
24  hours,  with  an  abundant  production  of  lactic  acid.  In  a  few  days  the  media 
become  so  acid  the  bacilli  are  killed  by  it. 

In  media  favorable  for  its  growth  bacillus  Bulgaricus  will  remain  alive  for 
more  than  9  months  if  kei  t  at  i5°C. 

129 


CHAPTER  XXXII 
BACILLUS  BOTULINUS 

Bacillus  botulinus  has  been  found  in  various  meat  products,  especially  sau- 
sage, less  frequently  in  eggs,  cheese,  butter  and  beans. 

Morphology. — Four  to  8  n  long,  0.5  to  i  ju  wide,  with  rounded  ends,  arranged 
singly,  in  groups  and  short  chains.  It  is  said  to  be  motile. 

Staining. — Stains  with  all  the  usual  stains  and  is  Gram  positive. 

Growth. — Bacillus  botulinus  is  an  obligate  anaerobic  organism  growing  best 
at  25°C.  to  30°C.  It  grows  best  in  meat,  especially  pork. 


FIG.  25. — BACILLUS  BOTULINUS. 
Some   individuals    containing    spores.     (After   van   Ermengen.} 

Media  containing  glucose  show  gas  formation,  gelatin  is  liquefied,  milk  is 
not  coagulated.  , 

Bacillus  botulinus  forms  spores  which  are  situated  near  the  extremity  of  the 
bacillus  and  are  slightly  wider  than  it,  causing  a  bulge.  Spores  are  killed  in  a 
short  time  at  8o°C.  in  a  moist  state.  Bacillus  botulinus  cannot  survive  in  brine 
containing  more  than  6  per  cent,  of  salt. 

Toxin. — Bacillus  botulinus  produces  a  powerful  extracellular  toxin  which 
accumulates  in  sausages,  meat  pies,  etc.,  containing  the  organism.  A  tempera- 
ture of  ioo°C.  destroys  this  toxin. 

Pathogenesis. — Bacillus  botulinus  does  not  infect  man,  but  when  food  con- 
taining its  toxin  is  eaten  the  toxin  is  absorbed  and  acts  as  a  powerful  poison, 
causing  death  in  25  per  cent,  of  cases.  Following  the  ingestion  of  toxin  a 
period  of  incubation,  never  less  than  6  hours,  elapses  before  the  onset  of 
symptoms.  Impairment  of  vision,  disturbance  of  speech,  increased  saliva- 

130 


BACILLUS  BOTULINUS  131 

tion,  general  muscular  weakness,  and,  in  severe  cases,  paralysis  are  constant 
signs. 

Postmortem  degeneration  of  ganglion  cells  of  the  anterior  horn  and  bulbar 
centers  is  found. 

A  period  of  incubation,  lasting  about  24  hours,  elapses  after  the  toxin  is  in- 
gested before  symptoms  develop. 

Diagnosis. — Bacteriological  diagnosis  can  only  be  made  by  finding  the  organ- 
ism or  its  toxin  in  food.  Cats  and  dogs  fed  on  meats,  etc.,  containing  the  toxin, 
or  injected  with  3  cc.  of  a  bouillon  culture  are  made  ill,  present  characteristic 
signs  and  frequently  die. 


CHAPTER  XXXIII 
BACILLUS  AEROGENES  CAPSULATUS 

B.  PERFRINGENS,  B.  EMPHYSEMATIS  VAGINAE,  B.  PHLEGMONIS  EMPHYSEMA- 

OR  B.  WELCHH 


Bacillatus  aerogenes  capsulatus  occurs  in  air,  dust,  soil  and  water;  it  is 
frequently,  if  not  constantly,  an  inhabitant  of  the  intestinal  canal  of  man  and 
animals. 

Extensive  studies  would  seem  to  indicate  that  this  organism  is  frequently 
present  in  the  gall-bladder,  liver  and  blood  of  apparently  healthy  normal  dogs 
and  in  the  liver  or  blood  of  some  apparently  healthy  people.  It  is  not  uncom- 
mon to  find  the  bacillus  aerogenes  capsulatus  in  blood  cultures,  and  cultures 
from  diseased  joints  in  cases  of  rheumatism  and  arthritis  deformans,  even  when 
it  is  demonstrable  that  bacillus  aerogenes  capsulatus  is  not  the  cause  of  the 
arthritis. 

This  organism  is  frequently  found  in  water  polluted  with  sewage. 

Morphology.  —  B.  aerogenes  capsulatus  is  a  large  bacillus,  3  to  8  ^  long,  has 
ends  which  are  square  or  slightly  rounded,  is  non-motile  and  is  arranged  singly 
and  in  chains.  Organisms  removed  from  tissue,  especially  blood,  usually  show 
a  distinct  capsule;  in  culture  the  capsule  may  disappear. 

Staining.  —  It  stains  with  all  the  common  stains  and  is  Gram  positive. 

Growth.  —  Bacillus  aerogenes  capsulatus  is  an  obligate  anaerobe  and  grows 
best  at  37°C. 

Bouillon  incubated  at  37°C.  shows  marked  cloudiness  in  12  to  24  hours; 
later  an  abundant  whitish  flocculent  sediment  forms. 

Agar.  —  Within  1  8  to  24  hours  round,  grayish  colonies  develop.  They  vary 
in  size  from  2  millimeters  to  i  centimeter  in  diameter,  are  flat  and  have  irregular 
margins. 

Stab  cultures  in  agar  show  gas  formation. 

Gelatin.  —  Growth  on  this  medium  is  the  same  as  on  agar.  Gelatine  is 
slowly  liquefied  in  the  majority  of  cases;  occasionally  liquefaction  does  not 
occur. 

Milk  is  rapidly  acidulated  and  coagulated. 

Potato.  —  Slight  growth  without  any  characteristic  appearance  occurs  on 
potato. 

Glucose,  lactose  and  saccharose   are  fermented;   gas   is  produced  from 
roteids. 

Bacillus  aerogenes  capsulatus  forms  spores. 

Toxin.  —  There  is  little,  if  any,  toxin  production. 

Resistance.  —  Bacillus  aerogenes  capsulatus  is  somewhat  more  resistant  to 
chemical  and  thermal  germicides  than  the  typhoid  bacillus.  Spores  are  highly 
resistant  to  chemicals  and  require  boiling  for  i  hour  to  kill. 

132 


BACILLUS   AEROGENES   CAPSULATUS  133 

Pathogenesis. — Bacillus  aerogels  capsulatus  may  cause  emphysematous 
gangrene  when  it  gains  entrance  into  body  tissue  at  the  site  of  an  injury.  It 
probably  is  capable  of  producing  gastro-intestinal  disturbances.  Relatively, 
the  pathogenic  power  of  bacillus  aerogenes  is  slight;  several  cubic  centimeters  of 
a  bouillon  culture  injected  into  the  circulation  of  a  rabbit  frequently  fails  to 
produce  ill  effect.  Guinea-pigs  seem  most  susceptible,  yet  some  strains  are 
not  pathogenic  for  these  animals. 

Diagnosis. — The  morphology,  and  character  of  growth  in  agar  and  gelatin 
stab  cultures,  is  usually  sufficient  to  establish  the  identity  of  this  organism. 
When  further  studies  are  desirable  and  when  the  bacillus  is  found,  associated 
with  other  organisms  and  needs  be  isolated,  a  bouillon  culture  is  made.  Three 
to  5  cc.  of  an  i8-hour-old  culture  is  injected,  intravenously,  into  a  rabbit.  About 
15  minutes  after  inoculation  the  rabbit  is  killed  with  ether  or  chloroform  and 
placed  in  an  incubator  at  37°C.  Twelve  to  18  hours  later  the  rabbit  will  appear 
much  distended  with  gas.  Crepitation  is  felt  in  the  subcutaneous  tissue  due 
to  accumulation  of  gas.  If  the  peritoneal  cavity  is  opened  by  puncture  and  a 
flame  touched  to  the  escaping  gas  it  will  burn  with  a  blue  flame.  Cultures  made 
from  the  blood,  liver,  heart  and  other  organs  will  yield  pure  cultures  of  the 
bacillus  aerogenes  capsulatus. 


CHAPTER  XXXIV 

BACILLUS  MALIGNI   (EDEMATIS 
BACILLUS  OF  MALIGNANT  EDEMA  OR  VIBRION  SEPTIQUE 

The  bacillus  of  malignant  edema  is  commonly  present  in  garden  soil, 
barnyard  manure  and  in  river  silt. 

Morphology. — It  is  a  large  bacillus,  3  to  10  p  long,  0.5  to  i.o  ju  wide,  has 
slightly  rounded  ends,  occurs  singly  and  in  chains  and  under  anaerobic  conditions 
is  slightly  motile. 

Staining  occurs  readily  with  the  usual  anilin  dyes  and  it  is  Gram  positive. 

Growth. — The  bacillus  of  malignant  edema  is  an  obligate  anaerobe  and 
grows  at  temperatures  between  i5°C.  and  4i°C.,  best  at  37°C. 

Bouillon  shows  cloudiness  in  12  to  18  hours;  after  24  to  48  hours  a  heavy, 
whitish  sediment  forms  and  the  bouillon  above  becomes  clear. 

Agar. — Colonies  develop  on  the  surface-  in  several  days;  they  are  small, 
round,  elevated  and  grayish. 

Stab  Cultures  in  Agar  show  growth  in  i  or  2  days.  It  appears  at  first  as 
a  white  line  along  the  stab  and  then  extends .  laterally,  giving  the  medium  a 
grayish  cloudy  appearance  and  forming  gas,  which  splits  the  agar. 

Gelatin. — Growth  on  gelatin  has  the  same  appearance  as  on  agar.  The 
medium  is  rapidly  liquefied. 

Blood  serum  is  liquefied. 

Milk  is  slowly  coagulated. 

Potato  shows  slight,  if  any,  growth. 

The  bacillus  of  malignant  edema  produces  indol  and  forms  spores. 

Resistance. — Bacilli  are  destroyed  in  a  moist  state  in  less  than  an  hour  at 
•7o°C.     They  are  more  resistant  to  chemical  germicides  than  the  typhoid  bacillus. 
Spores  are  very  resistant  to  all  germicidal  agents  and  require  boiling  for  at  least 
i  hour  to  kill  them. 

Toxin. — The  bacillus  of  malignant  edema  forms  a  small  amount  of  a  weak 
intracellular  toxin. 

Agglutinins. — Specific  agglutinins  occur  in  the  blood  of  immunized  animals. 

Pathogenesis. — This  organism  rarely  infects  man.  Being  an  obligate 
anaerobe  infection  is  most  apt  to  occur  in  puncture  and  deep  lacerated  wounds 
and  has  been  most  frequently  observed  in  severe  compound  fracture  wounds 
grossly  contaminated  with  dirt. 

Most  of  the  domestic  and  laboratory  animals  are  susceptible  to  the  bacillus 
of  malignant  edema. 

134 


BACILLUS   MALIGNI   CEDEMATIS  135 

Localized  edema  at  the  point  of  inoculation,  grave  septicemia,  convulsions 
and  death  are  observed  in  all  animals  subject  to  infection. 

Diagnosis. — The  bacillus  of  malignant  edema  is  easily  recognized  by'  in- 
jecting a  small  amount  of  fluid  suspected  of  containing  the  organism  into  a 
guinea-pig,  subcutaneously,  noting  the  effect  and  making  microscopic  and 
cultural  examinations  of  bacteria  found  in  the  blood  or  cedematous  area  when 
the  pig  dies. 


CHAPTER  XXXV 

• 

BACILLUS  TETANI 
(TACK  BACILLUS) 

The  bacillus  tetani  is  frequently  present  in  dust  and  soil,  especially  dust 
accumulated  in  old  buildings,  farms  and  cattle  sheds  and  in  soil  enriched  with 
horse  and  cow  manure. 

It  is  almost  constantly  present  in  the  intestinal  contents  and  f eces  of  sheep — 
the  source  of  cat-gut  used  in  surgery. 

The  comparative  frequency  with  which  tetanus  has  followed  black  powder 
burns,  would  seem  to  indicate  some  relation  between  this  substance  and  bacillus 
tetani. 

Morphology. — It  is  a  straight  bacillus  3  to  5  ju  long,  0.3  to  0.6  ju  wide,  has 
rounded  ends,  is  slightly  motile  and  produces  spores.  While  most  spore- 
bearing  bacteria  carry  their  spores  in  or  close  to  the  middle,  the  spores  occur 
in  tetanus  bacilli  at  one  end.  As  they  are  somewhat  larger  in  diameter  than  the 
bacillus  itself  and  cause  a  bulging,  a  characteristic  tack-like  appearance  is 
produced.  Tetanus  bacilli  are  arranged  singly  and  in  irregular  clumps  of  two, 
three  or  four  organisms. 

Staining. — Bacillus  tetani  stains  with  all  the  usual  anilin  stains  and  is 
Gram  positive. 

Growth. — Bacillus  tetani  is  primarily  an  obligate  anaerobe;  under  certain 
conditions  it  is  also  facultative  aerobic.  Growth  occurs  at  temperatures 
between  i$°C.  and  44°C.,  best  at  37°C. 

Bouillon  incubated  at  37°C.  under  anaerobic  conditions  becomes  cloudy  in 
1 8  to  36  hours;  later  a  grayish  sediment  forms  and  after  a  week  or  two  the 
medium  becomes  clear.  An  offensive  odor,  suggestive  of  burnt  horn,  is 
produced. 

Agar. — Small,  spherical,  whitish  colonies  appear  in  36  to  48  hours.  They 
have  a  dense  center  surrounded  by  a  thin,  irregular  zone. 

Stab  cultures  show  a  cloudy  appearance  of  the  medium,  which  is  split  by 
gas  formation. 

Gelatin. — Surface  growth  is  similar  to  that  observed  on  agar. 

Stab  cultures  in  gelatin,  as  in  agar,  at  first  show  fine  white  lines  of  growth 
extending  at  right  angles  to  the  stab ;  as  these  increase  the  medium  becomes 
cloudy.  Gelatin  is  slowly  liquefied,  and  when  liquefaction  is  complete  a  whitish 
sediment  is  deposited  and  the  medium  becomes  clear. 

Blood  serum  shows  nothing  characteristic  and  is  not  liquefied. 

Potato. — The  growth  of  the  tetanus  bacillus  on  potato  is  slight,  glistening 
and  colorless. 

Milk  is  not  coagulated. 

136 


BACILLUS   TETANI  137 

Bacillus  tetani  produces  indol,  carbon  dioxide  and  hydrogen  sulphide. 

Resistance. — Bacillus  tetani  is  slightly  more  resistant  to  germicidal  agents 
than  the  typhoid  bacillus,  but  its  spores  are  very  resistant.  They  survive  in 
5  per  cent,  phenol  and  i  :  1000  bichloride  solutions  at  20°C.  for  hours.  Steril- 
ization in  a  hot-air  sterilizer  sometimes  requires  a  temperature  of  i5o°C.  for 
several  hours.  Boiling  is  said  to  destroy  tetanus  spores  in  10  minutes,  but 
frequently  boiling  for  an  hour  or  more  is  necessary.  Steam  at  15  pounds 
pressure  destroys  them  in  a  few  minutes. 

Direct  exposure  to  sunlight  and  air  rapidly  alters  tetanus  spores  and  in  the 
course  of  10  to  15  days  destroys  them.  Spores  upon  the  surface  of  the  ground 
where  air  and  light  strike  them  die  in  one  or  several  weeks.  Upon  wood,  in  the 
dust  and  crevices  of  buildings,  where  they  are  protected  from  light  and,  to  some 
degree  from  air,  they  remain  viable  and  virulent  for  years. 

Toxin. — Bacillus  tetani  produces  a  powerful  extracellular  toxin.  The  symp- 
toms and  fatalities  of  tetanus  are  caused  by  the  filterable  toxin  called  tetano- 
spasmin.  It  seems  to  have  an  especial  affinity  for  nerve  tissues  and  to  travel 
to  the  central  nervous  system  along  the  motor  tracts. 

"  Tetanolysin "  is  a  separate  extracellular  toxin  produced  by  the  tetanus 
bacillus.  It  causes  hemolysis  of  the  red  blood  cells  of  many  animals. 

By  repeated  injection  of  sublethal  doses  of  toxin,  animals  can  be  immunized 
against  tetanus  and  the  serum  of  such  animals  is  highly  antitoxic. 

Agglutinins  are  not  present  in  the  blood  of  those  infected  with  bacillus 
tetani. 

Pathogenesis. — Tetanus  is  a  disease  having  a  high  mortality.  Man  and. 
the  horse  are  especially  susceptible;  guinea-pigs,  mice  and  rats  are  very  sus- 
ceptible, rabbits  less  so;  dogs  possess  a  considerable  degree  of  immunity. 

Absence  of  oxygen,  devitalization  of  surrounding  tissue  and  presence  of  other 
organisms  are  conditions  most  favorable  to  infection,  hence  the  disease  is  usually 
seen  following  puncture  wounds,  extensive  traumatism  and  deep  lacerations 
grossly  soiled  with  street  dust,  soil  or  cinders.  Bacillus  tetani  lodges  at  the 
point  of  entry;  it  does  not  enter  the  blood-stream  or  lymph-stream  and  does  not 
pass  to  other  parts  of  the  body.  The  invading  organisms  are  strictly  localized 
and  the  extracellular  toxin  they  liberate  travels  along  the  course  of  the  motor 
nerves  to  the  motor  centers. 

It  has  been  fairly  well  established  that  tetanus  spores  may  enter  the  body 
and  remain  dormant  at  the  atrium  of  infection  for  weeks,  eventually  being 
destroyed  or  removed  from  the  body  through  the  action  of  natural  /protective 
forces  or  as  the  result  of  injections  of  tetanus  antitoxin — or,  after  remaining 
dormant  for  a  number  of  days  or  weeks,  become  active,  producing  bacilli, 
toxin  and  the  characteristic  phenomena  of  tetanus.  Such  activity  after  quies- 
cence is  favored  by  a  local  or  general  weakening  of  the  natural  or  normal  im- 
munizing forces  as  commonly  follows  trauma,  intercurrent  infections  and 
certain  chemical  intoxications.  It  has  been  known  for  a  long  time  that  quinine 
is  an  activator  of  tetanus  spores  and  bacilli;  that  the  administration  of  small  or 
large  doses  of  quinine  favors  the  development  of  tetanus. 


138  MEDICAL  BACTERIOLOGY 

Diagnosis. — For  the  bacteriological  diagnosis  of  tetanus,  pus  or  fluid  is 
removed  from  any  wound  that  may  exist,  smears  are  made,  fixed,  stained  and 
examined  microscopically;  tetanus  bacilli  may  or  may  not  be  observed.  Agar 
and  bouillon  are  inoculated  with  the  pus,  two  tubes  of  each;  half  of  these  are 
incubated  under  aerobic  and  the  others  under  anaerobic  conditions  at  37°C. 
for  24  to  48  hou!rs  and  then  inspected  with  the  naked  eye  and  smears  made  for 
microscopic  examination. 

Such  examinations  not  infrequently  fail  to  disclose  tetanus  bacilli  or  spores 
that  are  present  and,  therefore,  bacteriological  examinations  are  not  depended 
upon  in  making  a  diagnosis  of  this  infection. 


CHAPTER  XXXVI 
BACILLUS  ANTHRACIS 

Bacillus  anthracis  occurs  in  soil  and  on  grasses  of  pasture  lands  traversed  by 
infected  animals  and  land  polluted  by  the  burial  of  animals  dying  with  anthrax. 
It  also  occurs  upon  wool  and  hides  of  animals  coming  from  districts  where  the 
disease  prevails. 

Morphology. — Bacillus  anthracis  is  a  large,  straight  non-motile  rod  with 
square  ends,  5  to  10  JJL  long  and  i  to  2  ju,  wide. 

Bacilli  observed  in  tissue  are  arranged  singly  and  in  pairs,  end  to  end,  and  do 
not  contain  spores.  Those  obtained  from  cultures  are  arranged  in  long  fila- 
ments and  show  spore  formation.  The  spores  form  in  the  middle  of  the  bacilli. 


FIG.  26. — BACILLUS  ANTHRACIS. 
Some  showing  spores.      (4  X  eyepiece  and  Ma  oil  immersion  objective.) 

Staining. — Bacillus  anthracis  stains  readily  with  the  usual  stains  and  is 
Gram  positive.  The  spores  require  special  methods  to  tint  them. 

Growth. — Bacillus  anthracis  is  an  aerobic  and  facultative  anaerobic  organ- 
ism. It  grows  at  temperatures  between  i5°C.  and  45°C.,  best  at  37°C. 

Bouillon  incubated  at  37°C.  shows  growth  in  24  hours,  a  whitish  pellicle 
forms  on  the  surface  and  a  stringy,  whitish  sediment  collects  in  the  bottom. 
When  at  rest  the  medium  remains  clear. 

Agar. — Round,  irregular-edged,  flat,  opaque,  whitish  colonies  appear  in  18 
to  36  hours  and  tend  to  coalesce,  forming  a  pellicle  covering  the  surface. 

Gelatin. — On  the  surface  of  gelatin  growth  appears  as  on  agar. 

Gelatin  stabs  show  growth  along  and  radiating  from  the  stab  described  as 
"inverted  fir-tree  growth."  Gelatin  is  slowly  liquefied. 

Potato. — A  dry,  whitish  pellicle  forms  on  the  surface. 

139 


140  MEDICAL  BACTERIOLOGY 

Milk  is  acidulated  and  coagulated. 

Resistance. — Bacillus  anthracis  is  only  slightly  more  resistant  to  heat  and 
chemical  germicides  than  the  typhoid  bacillus. 

The  spores  are  very  resistant  to  all  germicidal  agents;  exposure  to  strong 
chemicals,  direct  sunlight  and  drying  is  withstood  for  a  long  time.  'An  expo- 
sure of  several  hours  at  i5o°C.  to  20o°C.  is  required  to  destroy  spores  in  a  hot- 
air  sterilizer;  boiling  for  i  hour  sometimes  fails  to  kill  them;  steam  under  15 
pounds  pressure  destroys  anthrax  spores  in  a  few  minutes. 

Toxin. — Bacillus  anthracis  produces  no  extracellular  toxin. 

Agglutinins  and  lysins  are  not  demonstrable  in  the  blood  of  either  infected 
or  immunized  animals. 

Pathogenesis. — Many  domestic  animals  are  very  susceptible  to  anthrax, 
usually  contracting  the  infection  through  eating  from  pastures  contaminated 
with  anthrax  spores.  The  feces  from  such  animals  contains  spores.  Minute 
amounts  of  bacillus  anthracis  or  spores  injected  into  rabbits  or  guinea-pigs 
produce  death. 

In  man  infection  is  almost  entirely  confined  to  those  engaged  in  handling 
hides,  wool  or  cattle. 

Wool  sorters  are  usually  infected  through  the  respiratory  tract  and  suffer 
pulmonic  involvement,  followed,  in  most  cases,  by  bacteremia  and  death. 
Those  who  handle  hides  are  usually  infected  through  abrasions  of  the  skin,  a 
lesion  known  as  malignant  pustule  developing  at  the  point  of  inoculation.  The 
infection  may  remain  localized  or  the  bacteria  may  enter  the  blood-stream. 
The  mortality  is  high — 25  per  cent,  or  more. 

Infection  through  the  alimentary  canal  in  man  is  exceedingly  rare. 

Diagnosis. — When  the  disease  is  localized  bacteria  may  be  obtained  from  the 
pustule  and  adjacent  tissue;  if  bacteremia  occurs  organisms  may  be  obtained 
from  any  blood-vessels — even  capillaries  are  clogged  with  them. 

Bacillus  anthracis  is  easily  identified  by  its  morphology  and  growth  on  plain 
agar,  gelatin  or  bouillon. 


CHAPTER  XXXVII 

BACILLUS  SUBTILIS 

(HAY  BACILLUS) 

Bacillus  subtilis  is  present  in  soil,  dust,  air  and  almost  constantly  upon  hay. 
It  is  not  a  pathogenic  organism,  but  may  enter  chronic,  neglected  wounds  or 
ulcers  and  exist  there  as  a  saprophyte.  Frequently  is  it  found  in  contaminated 
milk,  blood  serum,  infusions  and  laboratory  culture  media.  It  is  a  spore-form- 
ing bacillus  and  these  spores  are  very  resistant,  withstanding  boiling  for  5  or  10 
minutes. 

Morphology. — Bacillus  subtilis  is  from  4  to  8  ju  by  about  0.7  /z,  has  rounded 


FIG.  27. — BACILLUS  SUBTILIS.     STAINED  BY  GRAM'S  METHOD. 
(4  X  eyepiece  and  Ma  oil  immersion  objective.) 

-ends,  is  slightly  motile,  carries  spores  near,  but  not  exactly,  in  the  middle,  and 
is  arranged  singly,  in  pairs  and  in  long  filaments. 

Staining. — It  stains  readily  with  all  the  usual  anilin  stains  and  is  Gram 
positive. 

Growth.' — Bacillus  subtilis  is  aerobic,  and,  to  a  degree,  anaerobic.  It  grows 
well  between  2o°C.  and  37°C.  on  all  culture  media,  forming  a  whitish  sediment 
in  fluid  media  and  forming  on  solid  media  round,  irregular-edged,  whitish 
colonies  which  tend  to  coalesce.  Gelatin  is  liquefied. 

To  insure  sterilization  by  hot  air  a  temperature  of  i5o°C.  must  be  maintained 
for  several  hours. 

141 


142  MEDICAL  BACTERIOLOGY 

Boiling  for  i  hour,  or  20  minutes,  in  an  autoclave  at  15  pounds  pressure 
kills  bacillus  subtilis  spores. 

Four  or  five  daily  exposures  at  8o°C.  for  10  minutes  is  sometimes  required 
when  sterilization  is  to  be  accomplished  by  the  fractional  or  intermittent  method. 

When  a  pure  culture  of  bacillus  subtilis  is  desired  for  study  and  none  is  at 
hand,  some  hay  dust  is  placed  in  sterile  water,  gradually  heated  to  the  boiling 
point  and  then  boiled  for  20  minutes. 

If  tubes  of  agar  are  planted  with  several  loopsful  of  this  decoction — imme- 
diately before  heating,  when  it  reaches  6o°C.,  8o°C.  and  ioo°C.,  and  again  when 
it  has  boiled  for  i  minute,  2  minutes,  5  minutes,  10  minutes,  15  minutes  and  20 
minutes — a  pure  culture  of  subtilis  will  be  obtained  and  a  study  of  the  culture 
made  before  and  after  exposure  to  different  degrees  of  heating  will  show  the 
variations  in  the  heat-resisting  power  and  different  bacteria  and  spores. 


CHAPTER  XXXVIII 
BACILLUS  PRODIGIOSUS 

Bacillus  prodigiosus  occurs  in  soil,  dust  and  water,  and  upon  exposed  food- 
stuffs, especially  those  containing  starch. 

Morphology. — Bacillus  prodigiosus  is  smaller  than  the  typhoid  bacillus, 
but  the  difference  is  not  sufficient  to  permit  differentiation.  It  is  motile  and 
possesses  from  six  to  eight  lateral  flagella.  It  is  arranged  singly,  in  pairs,  short 
filaments  and  in  irregular  groups. 

Staining. — It  stains  readily  with  all  the  anilin  dyes  and  is  Gram  negative. 

Growth. — Bacillus  prodigiosus  is  aerobic  and  facultative  anaerobic.  Under 
aerobic  conditions  it  produces  a  red  pigment;  this  does  not  occur  in  an  anaerobic 
state.  It  grows  best  between  2o°C.  and  25°C.;  above  37.5°^  pigment  is  not 
formed. 

Bouillon  becomes  cloudy  in  24  to  48  hours;  under  favorable  circumstances 
pigment  forms  and  gives  the  medium  a  reddish  tint;  after  several  days  a  sediment 
forms. 

Agar. — Growth  appears  in  1 8  to  24  hours,  as  round,  moist,  glistening,  whitish, 
pin-head-sized  colonies,  which  later  coalesce  and  cover  the  surface  with  a  red 
pellicle. 

Gelatin  is  liquefied  and  a  reddish  sediment  forms. 

Milk  is  acidulated  and  coagulated. 

Potato  is  the  best  medium  upon  which  to  observe  pigment  formation. 
Growth  covers  the  entire  surface  in  24  to  36  hours.  At  first  it  is  bright  red;  as 
the  culture  ages  the  pigment  darkens  until  it  becomes  brown. 

Glucose  is  not  acidulated,  but  occasionally  gas  is  formed.  Indol  production 
is  variable.  Bacillus  prodigiosus  does  not  form  spores. 

Resistance. — Bacillus  prodigiosus  is  sterilized  by  an  exposure  of  J£  hour  to 
i5o°C.  dry  heat,  or  J^  hour  at  8o°C.  moist  heat;  boiling  kills  it  immediately. 
It  is  slightly  more  resistant  to  chemical  germicides  than  the  typhoid  bacillus. 

Toxin. — Very  old  cultures  are  said  to  contain  toxin. 

Agglutinins. — Specific  agglutinins  are  present  in  the  serum  of  immunized 
animals. 

Pathogenesis. — Bacillus  prodigiosus  is  a  saprophyte,  in  old  cultures  it  is 
occasionally  slightly  pathogenic.  This  organism  is  of  interest  to  physicians 
because  it  is  frequently  present  in  dirty  foods  and  may  alter  them  in  a  deleterious 
manner;  also  it  is  used  in  preparing  Coley's  fluid,  an  agent  used  in  the  treatment 
of  certain  cases  of  sarcoma. 

143 


CHAPTER  XXXIX 
BACILLUS  PESTIS 

Bacillus  pestis  is  present  in  the  lymphatic  glands  and  in  the  blood  of  in- 
fected animals  and  man;  in  the  sputum  of  those  suffering  from  the  pneumonic 
form  of  the  disease,  in  the  carcasses  of  those  dead  of  plague  and  in  fleas  which 
have  fed  upon  infected  rats.  It  has  been  found  several  times  in  the  soil  of 
districts  where  plague  is  endemic. 

Morphology. — Bacillus  pestis  is  subject  to  marked  variations  in  size  and 


FIG.  28. — BACILLUS  PESTIS. 

Upper  half  of  field  shows  polar  staining.     Lower  half  of  field  shows  solid  staining. 
(4  X  eyepiece  and  Ma  oil  immersion  objective.) 

shape.  Bacilli  removed  from  the  lymphatic  glands  of  those  suffering  with 
plague  are  about  i  /z  to  2  ju  have  decidedly  rounded  ends,  stain  deeply  at  each 
end  and  show  a  clear  band  in  the  middle.  They  are  arranged  singly.  Young 
(24  to  48  hours)  cultures  in  bouillon,  agar  and  salt-agar  have  a  different  appear- 
ance; bacilli  from  bouillon  stain  solidly,  are  about  i  JJL  by  2  /x  and  are  arranged 
in  pairs,  end  to  end,  and  in  filaments,  as  well  as  singly.  Those  from  agar  and 
salt-agar  have  the  same  appearance,  but  do  not  show  filaments  as  in  bouillon. 

Old  agar  and  salt-agar  cultures  (5  to  6  days  old)  show  marked  involution 
forms,  bacilli  3  or  4  ju  long  and  i  ju  wide  showing  polar  staining,  and  larger  bacilli 
which  stain  solidly  and  are  curved  or  club-shaped.  Similar  involution  forms 
may  be  found  in  tissue.  Bacillus  pestis  is  not  motile. 

Staining. — B-acillus  pestis  stains  with  all  the  basic  anilin  dyes  and  is  Gram 
negative. 

144 


BACILLUS    PESTIS  145 

Growth. — Bacillus  pestis  is  an  aerobe  and  grows  best  at  temperatures 
between  3o°C.  and  38°C. 

Bouillon  growth  appears  as  a  whitish  sediment,  which  sticks  to  the  sides  or 
falls  to  the  bottom  of  the  container.  The  medium  usually  remains  clear, 
occasionally  it  becomes  turbid;  slight  pellicle  formation  may  occur. 

Bouillon  to  which  sufficient  sterile  oil  or  butter  has  been  added  to  form 
globules  on  the  surface,  if  kept  at  rest  during  incubation,  has  a  stalactite  forma- 
tion if  bacteria  develop.  Around  each  globule  these  stalactites  are  whitish 
and  when  the  medium  is  agitated  they  fall  to  the  bottom. 

Agar,  after  incubation  at  37°C.  for  24  hours,  shows  small,  round,  irregular- 
edged,  transparent  whitish  colonies.  Subcultures  show  more  abundant  growth 
than  those  made  from  tissue. 

Salt-agar  is  especially  valuable  to  demonstrate  involution  forms. 

Gelatin  surface  cultures  show  round  yellowish  colonies. 

Gelatin  stab  cultures  show  a  fine  thread-like  whitish  growth  along  the  stab 
with  a  yellowish  growth  at  the  surface.  Gelatin  is  not  liquefied. 

Blood  serum  growth  appears  as  on  agar. 

Milk  is  not  coagulated;  there  may  be  slight  acidulation. 

Potato  shows  slight,  if  any  growth  without  any  distinctive  feature. 

Bacillus  pestis  forms  acid,  but  no  gas  in  glucose;  there  is  neither  acid  nor 
gas  production  in  lactose  and  saccharose.  Spores  are  not  formed.  Indol  is 
not  formed. 

Resistance. — Exposure  to  direct  sunlight  will  kill  the  bacillus  pestis  in 
several  hours.  In  dried  pus  it  remains  alive  for  weeks.  It  has  been  found  alive 
in  soil,  protected  from  light,  after  several  months  and  in  water  after  i  month. 
It  remains  alive  and  virulent  in  the  bodies  of  those  dead  of  plague  for  weeks; 
when  putrefaction  occurs  bacillus  pestis  is  said  to  disappear  in  15  to  30  days. 
Five  per  cent,  carbolic  acid  solution  kills  it  in  10  minutes,  so  does  i  :  1000 
bichloride.  Hot-air  sterilization  requires  an  exposure  of  J^  hour  at  i5o°C.; 
in  a  moist  state  6o°C.  for  i  hour.  Boiling  kills  it  instantly. 

Toxin. — Bacillus  pestis  produces  an  intracellular  toxin. 

Agglutinins. — The  occurrence  of  agglutinins  and  precipitins  in  the  blood  of 
infected  and  immunized  persons  and  animals  is  irregular  and  slight. 

Pathogenesis. — Plague  is  a  disease  said  to  occur  in  many  animals  other  than 
man  and  rats;  among  them  may  be  mentioned  squirrels  and  guinea-pigs,  rabbits, 
cats,  chickens  and  monkeys. 

Much  the  most  common  and  important  is  the  occurrence  of  the  disease  in 
rats  and  man.  The  disease  usually  prevails  in  rats  of  a  district  shortly  before 
the  occurrence  of  an  epidemic  among  the  people;  this  association  is  almost  a 
rule  and  the  agent  of  transmission  is  the  flea.  Fleas  which  bite  stricken  rats 
imbibe  the  bacilli  and  implant  them  upon  the  skin  of  people  whom  they  bite. 
Some  believe  the  flies  and  mosquitoes  may  play  a  minor  part  in  the  dissemination 
of  plague. 

Two  forms  of  the  disease  occur  in  man:  the  bubonic,  distinguished  by  the 
enlargement  of  many  or  all  of  the  superficial  lymph  glands,  with  or  without, 


146  MEDICAL  BACTERIOLOGY 

usually  without,  involvement  of  the  lungs,  and  the  pneumonic  form,  dis- 
tinguished by  the  occurrence  of  pneumonia  with  slight,  or  no  enlargement  of 
superficial  lymph  glands. 

In  both  the  bubonic  and  pneumonic  forms  of  the  disease,  bacteremia  is 
said  to  occur;  in  both  bacilli  are  present  in  the  lymph  glands  and  may  be  in  the 
urine. 

If  the  bubonic  form  is  complicated  with  pneumonia  bacilli  may  occur  in  the 
sputum.  In  the  pneumonic  form  bacilli  are  regularly  present  in  the  sputum  in 
large  numbers,  from  the  onset  throughout  the  disease. 

Diagnosis. — Microscopic  examination  of  the  sputum  in  pneumonic  cases 
discloses  the  cause.  » 

For  the  diagnosis  of  suspected  and  bubonic  cases  lymph  glands  are  searched 
for,  massaged  and  some  of  their  contents  removed  with  a  sterile  syringe  and 
needle.  A  portion  of  the  material  so  obtained  is  planted  on  salt-agar,  in  bouillon 
containing  oil  and  in  MacConkey's  bile-salt  medium;  about  Ko  to  }/£  cc.  is 
injected  subcutaneously  into  a  guinea-pig,  smears  are  made,  fixed  and  stained 
with  carbolfuchsin,  others  with  methylene  blue  and  still  others  by  Gram's 
method. 

The  discovery  of  a  polar  staining  cocco  bacillus  in  the  smears,  involution 
forms  on  salt-agar  after  24  hours  at  37°C.,  death  of  the  inoculated  animal  and 
recovery  from  its  body  of  organisms  identical  with  those  removed  from  the 
gland  establishes  the  diagnosis. 

When  examining  rats,  in  addition  to  making  blood  cultures,  and  subcultur- 
ing  these  on  glucose,  lactose,  saccharose  and  salt-agar,  careful  examination  of 
the  spleen  and  liver  should  be  made  to  detect  characteristic  changes,  produced 
by  plague. 


CHAPTER  XL 
BACILLUS  MALLEI 
(GLANDERS  BACILLUS) 

The  bacillus  mallei  is  present  in  the  nasal  discharge  and  saliva  of  many  of 
those  suffering  from  the  disease,  also  in  the  exudate  from  open  skin  lesions. 
From  such  sources  the  dust  and  soil  of  stables  and  horse  troughs  are  at  times 
polluted. 

Glanders  is  a  disease  of  horses,  asses  and  mules,  occasionally  carnivora  fed 
the  meat  of  animals  dead  of  the  disease  develop  glanders,  and  the  disease 
occasionally  afflicts  man. 

Infection  with  glanders  is  confined  almost  entirely  to  persons  in  frequent 
contact  with  horses. 

Morphology. — Bacillus  mallei  occurs  both  as  straight  and  slightly  curved, 
non-motile  rods,  3  ju  to  4  ju  long,  0.50  /z  to  0.75  p  wide,  having  rounded  ends, 
and  arranged  singly  in  irregular  masses.  Old  cultures  show  larger,  club-shaped, 
granular  forms,  long  (apparently  branched)  filamentous  forms,  and  chains  of 
small  coccoid  forms.  Bacillus  mallei  does  not  form  spores. 

Staining. — This  bacillus  stains  readily  with  all  the  common  stains  and  is 
Gram  negative.  It  can  be  decolorized,  after  staining,  by  washing  with  water, 
much  easier  than  other  bacteria.  As  a  rule,  staining  is  not  uniform,  many 
organisms  showing  an  oval  unstained  central  portion,  falsely  suggesting  the 
presence  of  spores. 

Growth. — Occurs  from  23°C.  to  4i°C.,  best  at  37°C.  Bacillus  mallei  is 
aerobic  and  only  slightly  facultative  anaerobic. 

Bouillon  incubated  at  37°C.  becomes  cloudy  in  24  hours,  later  a  tough, 
mucoid,  white  sediment  appears  and  in  several  days  a  white  pellicle  sometimes 
covers  the  surface. 

Agar  incubated  at  37°C.  shows  surface  growth  in  20  to  24  hours,  at  first  a 
white,  transparent  streak,  which  spreads,  thickens  and  becomes  opaque  as 
growth  continues. 

Glycerin  agar  favors  a  more  abundant  growth,  the  surface  usually  being 
covered  with  a  film  having  the  same  appearance  as  observed  on  plain  agar. 

Gelatin. — Scant,  almost  invisible  growth  occurs  upon  or  in  gelatin  after 
several  days,  it  is  not  liquefied. 

Potato,  neutral  or  nearly  neutral  in  reaction  is  an  important  medium  in  the 
study  of  the  glanders  bacillus.  In  48  hours,  incubated  at  37°C.,  a  character- 
istic, moist,  thick,  yellowish  solid  growth  develops.  As  the  culture  ages  it 
turns  brown  and  the  potato  adjacent  to  it  becomes  black. 

Milk  is  acidulated  and  in  i  to  2  weeks  slowly  coagulates. 

i47 


148  MEDICAL  BACTERIOLOGY 

Resistance. — In  moist  state  bacillus  mallei  is  killed  in  i  hour  at  8o°C.,  in 
the  Arnold  steam  sterilizer  and  autoclave  it  is  destroyed  in  i  or  2  minutes. 
Thorough  drying  rapidly  attenuates  and  soon  kills  it.  In  the  hot-air  sterilizer 
it  is  killed  within  i  hour  at  i2o°C.  At  room  temperature,  5  per  cent,  phenol 
solution  kills  it  in  less  than  15  minutes;  1:1000  bichloride  of  mercury  in  less 
than  45  minutes.  Complete  exposure  to  direct  sunlight  will  kill  it  in  several 
days.  This  bacillus  has  been  found  alive  and  virulent  after  remaining  60  days 
in  a  water  trough. 

Toxin. — Bacillus  mallei  produces  an  intracellular  toxin,  mallein,  obtained 
the  same  as  tuberculin,  and  employed  in  the  diagnosis  of  glanders  in  animals, 
the  same  as  tuberculin  is  employed  in  the  diagnosis  of  tuberculosis  in  cattle; 
with  this  exception — at  present  mallein  is  usually  dropped  on  the  eye,  not 
injected  subcutaneously.  There  is  no  extracellular  toxin  production. 

Pathogenesis. — Infection  in  man  is  usually  through  an  abrasion  of  the 
skin,  a  nodule  developing  at  the  atrium  of  infection  is  rapidly  surrounded  by  a 
zone  of  intense  inflammation.  High  fever,  severe  malaise,  a  general  papular 
eruption  that  becomes  pustular  frequently  occurs  in  severe  acute  cases  which 
are  often  fatal  in  2  or  3  weeks. 

Less  acute  cases  and  those  terminating  in  recovery  may  not  develop  a 
generalized  eruption.  Infections  running  a  chronic  course  in  man  are  marked 
by  enlargement  of  the  lymphatic  glands  and  vessels  and  are  often  fatal. 

Rabbits  and  guinea-pigs  are  susceptible  to  inoculation. 

Diagnosis. — Laboratory  diagnosis  is  based  on  microscopical  and  cultural 
studies  of  the  exudate  from  open  lesions,  or  of  fluid  aspirated  from  enlarged 
glands,  or  excised  glands  emulsified  with  normal  salt  solution,  and  the  result 
of  guinea-pig  inoculation. 

The  exudate  from  open  lesions  usually  contains  organisms  in  addition  to  the 
Glanders  bacillus,  and  should  be  thoroughly  cleansed  and  covered  with  a  moist 
aseptic  dressing  for  several  hours  before  specimen  is  removed  for  examination. 
Suspected  material  is  planted  on  potato  medium. 

When  inoculation  test  is  to  be  made,  exudates  and  superficial  tissue  apt  to 
be  contaminated  with  other  organisms  are  injected  subcutaneously  into  a  guinea- 
pig.  When  nodules  develop  or  the  pig  dies,  under  strict  aseptic  precautions, 
a  nodule  is  excised,  placed  in  a  glass  mortar  with  several  times  its  volume  of 
normal  salt  solution  and  emulsified.  One  or  2  cc.  of  this  emulsion  is  injected 
into  the  peritoneal  cavity  of  a  male  guinea-pig. 

Subcutaneous  nodules  removed  from  patients  are  emulsified  and  injected 
directly  into  the  peritoneal  cavity  of  a  male  guinea-pig. 

When  injected  this  way,  in  pure  culture,  into  male  guinea-pigs,  bacillus 
mallei,  frequently,  but  not  always,  causes  a  severe  orchitis  with  enlargement 
and  suppuration. 


CHAPTER  XLI 

SPIROCILETA  OBERMEYERI 
(SPIROCHJETA,  OF  RELAPSING  FEVER) 

Spirochaeta  Obermeyeri  occurs  in  the  blood  of  those  suffering  with  relapsing 
fever  and  in  certain  lice,  bugs  and  ticks  that  have  fed  upon  infected  persons. 

Morphology. — Spirochaeta  Obermeyeri  show  marked  variations  in  length; 
the- average  dimensions  are  20  /z  long  by  i  n  wide.  It  is  spiral,  the  curves  being 
of  uneven  lengths  and  depth.  It  is  actively  motile  and  progresses  with  a  screw- 
like  motion.  The  ends  are  pointed  and  it  is  said  to  possess  terminal  flagella. 

Staining. — Same  as  for  treponema  pallidum  (see  page  150). 

Growth. — Cultivation  on  artificial  media  is  difficult;  most  attempts  result 
in  failure  and  it  is  not  necessary  for  diagnostic  purposes. 

Pathogenesis. — Inoculation  with  blood  taken  from  relapsing  fever  patients 
has  produced  the  disease  in  monkeys  and  rats.  Rabbits  and  guinea-pigs  are 
immune.  Spirochaeta  Obermeyeri  has  been  found  alive  and  infectious  in  ticks 
and  leeches  several  days  after  they  fed  upon  infected  persons.  When  the  body 
louse  imbibes  this  spirillum  it  may  pass  it  through  heredity  to  the  next 
generation. 

Diagnosis. — In  suspected  cases  blood  should  be  obtained  from  the  finger 
during  the  febrile  period.  Several  thin  films  should  be  made  on  clean  slides  or 
cover  glasses;  these  should  be  dried  at  room  temperature  or  placed  in  an  incu- 
bator, not  flamed.  At  the  same  time  a  drop  of  blood  should  be  examined  in 
the  fresh  state.  Place  it  on  a  clean  slide,  gently  drop  a  clean  cover  upon  it  and 
immediately  examine  with  an  oil  immersion  lens. 

The  organisms,  most  of  them  five  or  ten  times  as  long  as  the  diameter  of  a 
red  blood  cell,  are  observed  moving  about  with  great  rapidity,  by  a  screw-like 
and  also  undulatory  motion,  often  pushing  red  cells  aside. 

During  the  febrile  period  of  the  disease  Spirochaeta  Obermeyeri  retreats  from 
the  peripheral  blood  and  examination  at  this  time  is  not  advisable. 

Differentiation  of  the  closely  related  Spirochaeta  can  be  made  only  by 
agglutination  tests  with  the  serum  of  immunized  rats. 


149 


CHAPTER  XLII 


TREPONEMA  PALLIDUM 

Treponema  pallidum  is  present  in  the  ulcers  and  skin  lesions  of  syphilis,  most 
numerous  in  the  primary  and  secondary  period  of  the  disease,  scant  later  in  the 
infection.  In  small  numbers  they  are  present  in  the  blood  at  times  during 
the  disease  and  present  in  the  enlarged  glands  and  affected  organs. 

Morphology. — Treponema  pallidum  is  variable  in  length,  averaging  about 
'6  fj,  long  and  0.25  /*  in  diameter.  It  is  spiral,  showing  from  five  to  twenty  curves, 
which  are  fairly  uniform  in  depth  and  length;  they  are  deepest  at  the  center  and 

gradually  become  less  marked  toward  the 
ends,  just  like  a  corkscrew.  The  ends  are 
pointed  and  there  is  said  to  be  a  single  term- 
inal flagellum.  Treponema  pallidum  is 
actively  motile  and  progresses  with  a  screw- 
like  motion;  lateral  motion  is  less  marked. 
Reproduction  occurs  by  both  longitudinal 
and  transverse  division. 

Staining. — Ordinary  .bacterial  stains  fail 
to  or  only  poorly  stain  treponema  pallidum. 
The  best  method  of  staining  smears  is  the 
following: 


PALLIDUM. 


FIG.   ,  29. — TREPONEMA 
(Coplin.) 

Left  half,  Giemsa  stain,  spread 
from  congenital  syphilis;  right  half, 
Levaditi  stain  of  section  of  lung, 
congenital  syphilis;  this  (silver 
method)  makes  the  organisms  appear 
much  broader.  A,  Pairs  united  at 
one  end.  B,  Twisted  pairs.  C, 
Double  length,  apparently  dividing 
form. 


GIEMSA'S  METHOD 

1.  Make  a  thin,  even  smear,  dry  in  air 
without  heating. 

2.  Fix  in  absolute  alcohol  for  J^  hour. 

3.  Apply  the  following  stain  for  i  hour: 
Giemsa's  solution  (Griibler),  i  part. 

1  per  cent,  aqueous  sol.  sodium  carbonate,  i  part. 
Distilled  water,  i  part. 

After  staining,  wash  with  distilled  water,  dry  in  air  and  mount. 

The  stain  must  be  freshly  prepared  just  before  using. 

For  the  staining  of  treponema  in  tissue  the  following  is  most  satisfactory: 

LEVADITI'S  METHOD 

1.  Cut  the  fresh  tissue  into  small,  thin  pieces. 

2.  Fix  in  10  per  cent,  formalin  for  24  hours. 

3.  Harden  in  absolute  alcohol  for  24  hours. 

4.  Wash  in  distilled  water. 

5.  Place  in  following  solution,  freshly  prepared: 

2  per  cent,  aqueous  solution  of  silver  nitrate,  90  cc. 

150 


TREPONEMA   PALLIDUM  151 

Keep  in  this  bath  for  3  days  at  body  temperature,  excluding  light  and 
changing  the  solution  daily. 

6.  Place  in  the  following  solution  for  24  hours  at  room  temperature: 

5  per  cent,  solution  of  formaldehyde,  50  cc. 
Pyrogallic  acid,  i  Gm. 

7.  Wash  in  distilled  water. 

8.  Dehydrate  in  absolute  alcohol. 

9.  Clear  in  xylol. 

10.  Imbed  in  paraffin. 

11.  Section  and  mount. 

Treponema  appear  black. 

Growth. — Cultivation  of  treponema  pallidum  on  artificial  media  is  difficult 
and  time-consuming;  most  attempts  are  futile  and  as  yet  this  method  of  study 
is  not  practical  in  diagnosis. 

Material  taken  from  chancres  and  other  open  lesions  containing  treponema 
also  contains  numerous  other  organisms  and  isolation  of  the  treponema  from 
these  is  effected  by  inoculating  the  material  into  a  rabbit's  testicle. 

When  the  contaminating  organisms  die  out  and  the  treponema  multiply, 
media  planted  with  an  emulsion  of  such  a  testicle  yield  pure  cultures. 

The  treponema  pallidum  is  an  obligate  anaerobic  and  has  only  been  propa- 
gated in  ascitic  broth  and  ascitic  agar  containing  a  piece  of  sterile  rabbit 
testicle. 

Stab  cultures  in  deep  tubes  of  agar,  incubated  at  37°C.,  after  several  days, 
show  growth  near  the  bottom  of  the  tube  which  first  appears  as  fine  white  lines 
radiating  from  the  stab. 

The  character  of  lesion  that  develops  following  inoculation  of  a  rabbit 
depends  upon  the  tissue  in  which  the  treponema  are  deposited.  If  deposited 
in  the  subcutaneous  tissue  of  the  scrotum  a  chancre  usually  develops.  If  de- 
posited deep  in  the  testicular  tissue  gumma  develop.  The  production  of  gumma 
is  desired  when  rabbits  are  inoculated  as  a  primary  step  to  procure  pure  cultures, 
because  the  gumma  are  most  apt  to  contain  spirochaeta  not  associated  with 
other  organisms. 

Resistance. — Treponema  pallidum  is  more  susceptible  to  mercury  and 
salvarsan  than  to  other  chemical  germicides.  It  does  not  form  spores  and 
probably  is  destroyed  by  short  exposure  to  moderate  degrees  of  heat. 

The  time  it  can  remain  alive  and  infectious  on  linen,  glass,  soap  and  other 
objects,  subject  to  contamination  is  undetermined;  that  it  does  survive  for  short 
periods  at  least  on  such  objects  has  been  proved  by  infections  contracted  through 
contact  with  them. 

Pathogenesis. — Treponema  pallidum  occurs  in  man  only.  Conditions 
similar  to  syphilis  can  be  produced  in  monkeys  and  rabbits  by  inoculation. 
Syphilis,  like  some  other  diseases,  produces  a  hypersusceptibility  of  the  skin  to 
irritants  in  general  and  especially  to  the  infecting  organism.  In  syphilis  this 
phenomenon  is  most  marked  in  the  tertiary  period.  Noguchi  has  attempted  to 
base  a  diagnostic  test  on  this  fact — the  test  is  not  generally  available  on  account 


152  MEDICAL  BACTERIOLOGY 

of  the  difficulty  of  obtaining  pure  cultures  of  treponema  pallidum,  the  almost 
impossible  feat  of  differentiating  similar  spirochseta  from  the  treponema,  and 
the  difficulty  of  keeping  pure  cultures  from  contamination.  Toward  the  end 
of  the  primary  or  early  in  the  secondary  stage  of  syphilis  a  number  of  properties 
are  demonstrable  in  the  blood,  which  are  not,  practically  speaking,  found  in  the 
blood  of  healthy  or  diseased  persons  free  of  syphilis.  By  far  the  most  important 
of  these,  from  a  diagnostic  consideration,  are  amboceptors  or  complement  fixing 
.bodies.  These  are  nearly  always  demonstrable  when  the  disease  is  active  and 
are  also  demonstrable  in  the  majority  of  latent  cases.  To  detect  the  presence 
or  absence  of  these  antibodies  the  Wassermann  test  is  employed. 

Diagnosis. — In  the  first  stage  of  the  disease  ulcers  and  adjacent  enlarged 
glands  should  be  examined  for  treponema  and  a  Wassermann  test  made.  A 
negative  Wassermann  at  this  time  has  no  significance. 

In  the  second  stage  of  the  disease  a  Wassermann  test  should  be  made,  and, 
if  desired,  papules,  ulcers  and  enlarged  glands  adjacent  to  them  may  be  examined 
for  treponema. 

At  this  time  nearly  all  cases  of  syphilis  give  a  positive  Wassermann;  a  nega- 
tive is  suggestive  of  non-syphilitic  nature  of  the  malady. 

In  the  third  stage  of  the  disease  the  Wassermann  test  should  be  performed  as 
in  the  primary  and  secondary  stages,  and  if  negative  the  spinal  fluid  should  then 
be  examined,  because  at  this  time  a  positive  reaction  may  be  obtained  with  the 
spinal  fluid,  even  though  the  blood  is  negative. 

EXAMINATION  OF  ULCERS  FOR  TREPONEMA  PALLIDUM 

Wash  the  ulcer  free  of  extraneous  matter  with  sterile  normal  salt  solution. 
Avoid  bleeding  and  scrape  some  material  from  the  ulcer.  Place  a  small  por- 
tion of  the  scrapings  in  a  drop  of  salt  solution  on  a  slide,  gently  drop  a  cover 
glass  on  it  and  lute  with  vaseline  or  paraffin.  This  should  be  examined  with 
the  dark-field  illuminator,  or  if  that  is  not  available,  with  a  J^2  ou*  immer- 
sion objective  and  an  18  ocular. 

When  the  scrapings  are  obtained  several  slides  should  be  prepared  by  spread- 
ing thin,  even  films,  drying  them  in  air  or  in  an  incubator  at  37°C.,if fixing  in 
alcohol  and  staining  by  Giemsa's  method. 

If,  in  such  preparations,  an  organism  is  observed  that  appears  to  be  trepo- 
nema pallidum,  one  should  remember  that  a  number  of  organisms,  microscop- 
ically indistinguishable  from  treponema  pallidum,  may  be  found  in  superficial 
ulcers. 

Failure  to  find  treponema  pallidum  in  scrapings  from  ulcers  and  in  fluid  from 
papules  and  glands  does  not  indicate  the  absence  of  syphilis,  as  the  organisms 
are  irregularly  distributed  and  often  scant  in  the  material  taken  for  examination. 

EXAMINATION  OF  GLANDS 

Enlarged  glands  should  be  searched  for;  if  found,  they  are  massaged,  the 
overlying  skin  asepticized  and  some  of  the  glandular  contents  removed  with  a 
sterile  syringe  for  microscopic  examination.  When  material  so  obtained  con- 


TREPONEMA   PALLIDUM  153 

tains  motile  organisms  having  the  morphology  of  treponema  pallidum,  it  is 
strong,  one  might  say  conclusive,  evidence  of  syphilis,  as  contamination  is 
practically  precluded. 

EXAMINATION  OF  PAPULES 

The  admixture  of  blood  with  material  to  be  examined  is  avoided  by  pinching 
up  the  papule  with  a  hemostat,  nicking  the  skin  with  a  knife  and  obtaining  the 
serum  with  a  capillary  tube. 

Experience  has  shown  there  is  great  irregularity  in  the  distribution  of  tre- 
ponema in  tissues,  so  that  some  sections  of  an  organ  will  present  few  or  no  tre- 
ponema while  other  sections  of  the  same  organ  present  many  in  each  microscopic 
field. 

The  application  of  germicidal  washes  and  dressings,  especially  those  con- 
taining mercury,  to  open  syphilitic  lesions,  causes  a  disappearance  of  treponema 
and  attempts  to  firrd  organisms  in  scrapings  and  fluid  from  such  lesions  are 
usually  futile  until  after  such  washings  and  dressings  have  been  discontinued 
for  at  least  12,  better  24  hours. 

Subcutaneous  and  intracutaneous  injections  of  cocain,  and  other  methods 
of  producing  local  anesthesia,  usually  decreases  the  number  of  treponema  that 
may  be  found  in  syphilitic  lesions  or  causes  the  treponema  to  totally  disappear, 
hence  no  attempt  at  producing  local  anesthesia  should  precede  removal  of  fluid 
or  scrapings  from  ulcers,  papules  or  lymphatic  enlargements  for  examination 
with  the  dark  field  microscope  or  microscopic  examination  after  staining  and 
for  the  same  reason  no  anesthetic  should  be  applied  prior  to  excision  of  tissue 
to  be  examined  for  treponema. 

THE  WASSERMANN  TEST 

1.  Under  aseptic  precautions  at  least  5  cc.  of  blood  are  obtained  from  a 
prominent  vein  at  the  elbow  with  a  sterile  syringe;  the  blood  is  immediately 
transferred  to  a  sterile  tube  and  placed  in  an  ice  box  until  serum  separates  from 
the  clot. 

2.  The  serum  is  collected  and  heated  to  55°C.  for  J^  hour  in  a  water  bath. 

3.  The  patient's  serum  is  mixed  with  fresh  guinea-pig  serum  and  liver  extract 
and  incubated  at  37°  for  i  hour. 

4.  After  incubating  for  an  hour  red  blood  corpuscles  and  serum  of  a  rabbit, 
immunized  against  them,  is  placed  in  the  tube,  which  is  again  incubated  for 
an  hour. 

If  hemolysis  occurs  the  reaction  is  negative  (see  page  241). 


CHAPTER  XLIII 

SPIROCHJETA  PERTENUIS 
SPIROCH^ETA  REFRINGENS  AND  SPIROCHJETA  DENTIUM 

Spirochaeta  pertenuis  is  the  cause  of  a  non-venereal  tropical  skin  disease, 
called  "frambcesia  tropica,"  or  "yaws."  Spirochaeta  pertenuis  is  regularly 
found  in  the  papules  of  the  diseased  before  treatment.  It  has  the  same  size, 
shape  and  motility  as  the  treponema  pallidum  and  when  subjected  to  various 
methods  of  staining  reacts  just  as  the  treponema  does  except  that  as  a  rule 
spirochaeta  pertenuis  stains  more  readily.  Patients  infected  with  yaws  have  a 
positive  Wassermann  reaction  just  the  same  as  syphilitics  and  yaws  promptly 
answers  to  salvarsan  and  mercury. 

Some  observers  believe  there  is  a  close  relationship  between  the  treponema 
pallidum  and  spirochaeta  pertenuis. 

Diagnosis  is  made  by  pinching  one  or  several  of  the  skin  lesions  with  a 
hemostat,  nicking  it  with  a  scalpel  so  as  to  obtain  serum  and  not  blood.  The 
fluid  that  exudes  is  collected  in  a  capillary  tube,  smeared  on  cover  glasses,  dried, 
fixed  in  absolute  alcohol  for  15  minutes  and  stained  with  equal  parts  of  Giemsa's 
solution  and  water  for  20  minutes. 

The  large  number  of  distinctly  stained  spirochaeta  observed  in  every  cover- 
glass  preparation,  the  abundance  of  these  organisms  in  every  lesion,  of  a  fully 
developed  case  of  yaws,  untreated,  serves  to  establish  the  diagnosis  and  dif- 
ferentiate it  from  syphilis — the  lesions  of  which  do  not  so  uniformly  yield  fluid 
containing  demonstrable  treponema  and  almost  never  show  so  many  organisms. 

Spirochaeta  dentium  is  a  saprophyte  present  in  the  mouth,  particularly 
between  the  teeth.  It  is  microscopically  indistinguishable  from  the  treponema 
pallidum. 

Spirochaeta  refringens  has  been  found  in  ulcers  of  the  skin,  in  smegma  and  in 
the  mouth.  Usually  it  is  larger  and  more  easily  stained  than  the  treponema 
pallidum,  yet  microscopic  differentiation  is  at  times  impossible  and  often 
questionable. 


154 


CHAPTER  XLIV 
THE  HIGHER  BACTERIA 

Between  the  lower  forms  of  bacteria,  the  unicellular  rods,  spheres  and  spirals, 
which  reproduce  by  fission  only,  and  the  true  molds  which  have  differentiated 
parts,  comparable  to  the  roots,  trunk  and  fruit  of  trees,  there  are  intermediate 
forms  difficult  to  classify  satisfactorily.  Three  groups  in  this  class  are  of  in- 
terest to  the  medical  bacteriologist:  leptothrix,  cladothrix  and  strep  to  thrix; 
sometimes  referred  to  collectively  as  trichomycetes. 

LEPTOTHRICES 

Leptothrices  have  been  found  in  inflammatory  lesions  of  the  mouth;  they 
are  frequently  observed  in  saliva  and  sputum  from  healthy  persons,  more  often 
when  there  are  decaying  and  ill-cared-for  teeth. 

Morphology. — They  are  rod-shaped  and  vary  in  length  from  30  to  1 50  /*  and 
in  width  from  0.5  to  2  /z;  usually  they  are  seen  about  i  /x  wide  and  50  n  long.  Some 
are  straight,  others  curved  or  twisted,  and  they  are  arranged  singly,  in  filaments 
and  in  clumps.  Some  forms  are  segmented,  others  are  not. 

Staining. — Leptothrices  stain  readily  with  the  usual  aniline  dyes. 

Growth. — Attempts  to  cultivate  on  artificial  media  have  been  unsuccessful. 

Pathogenesis. — The  leptothrices,  in  all  probability,  are  non-pathogenic  and 
occur  in  the  mouth,  sputum  or  saliva  as  saprophytes. 

CLADOTHRICES 

Cladothrices  are  distinguished  from  leptothrices  and  streptothrices  by  false 
branching.  They  are,  therefore,  distinguished  from  leptothrices,  which  do  not 
show  branching.  Whether  or  not  reported  cases  of  cladothrix  infection  were 
such,  is  a  mooted  question,  as  differentiation  between  true  and  false  branching 
is  difficult  to  determine.  In  any  case,  cladothrix  infections  are  not  common 
if  they  do  occur  and  clinically  are  similar  to  streptothrix  infections. 

STREPTOTHRICES 

Streptothrices  are  distinguished  by  true  branching,  septate  filaments  and 
reproduction  by  conidia,  which  form  in  rows. 

Long  filaments  may  be  observed  singly  and  intertwined.  Some  appear 
chiefly  as  short,  stout  rods.  Frequently  both  short  and  long  filaments  terminate 
in  a  club-shaped  swelling. 

STREPTOTHRIX  ACTINOMYCOSIS 

Streptothrix  actinomycosis  occurs  upon  grain  and  pasture  land  contaminated 
with  the  discharges  from  open  lesions  of  cattle,  and  in  the  pus  and  sinus  dis- 
charges of  the  disease,  also  in  sputum  when  the  lungs  are  involved. 


156 


MEDICAL  BACTERIOLOGY 


Morphology. — Pus  or  fluid  discharging  from  an  actinomycotic  lesion  shows 
hard,  brittle,  irregular,  pin-point-sized,  yellowish  and  whitish  granules.  Place 
one  of  these  granules  on  a  slide,  in  a  drop  of  glycerin  and  crush  it  with  a  cover 
glass.  Microscopic  examination  will  reveal  rosette-like  masses.  The  dense 
centers  of  these  are  composed  of  interwoven  filaments,  and  the  outer  zone  of 
club-shaped  bodies  arranged  in  ray  form.  Here  and  there,  isolated  clubs  and 
filaments  are  observed,  occasionally  a  filament  terminating  in  a  club-shaped 
body.  The  filaments  vary  in  length  from  5  to  15  /*,  the  clubs  are  about  25  to 
10  /*.  The  addition  of  a  drop  of  20  per  cent,  potassium  hydrate,  or  a  drop  of 
Gram's  solution,  makes  them  appear  more  distinctly.  These  granules  are  not 


FlG.    30. ACTINOMYCES,  SHOWING  BRANCHING  OF  THE  FILAMENTS.       STAINED  WITH  FUCHSIN. 

(4  X  eyepiece  and  M2  oil  immersion  objective.) 

always  present.  Early  in  the  disease,  when  resistance  to  infection  is  slight,  the 
pus  may  only  contain  filaments.  The  filaments  are  long  and  short  and  show 
branching. 

They  stain  with  all  the  usual  anilin  dyes  and  are  Gram  positive  and  are  acid- 
fast  (to  a  lesser  degree  than  tubercle  bacilli),  but  are  not  alcohol-fast. 

Organisms  removed  from  culture  media  appear  differently.  Young  cultures 
show  fine,  branching,  non-septate  filaments,  some  flf  which  are  very  long.  Older 
cultures,  that  have  developed  aerial  hyphae,  show  short,  thick,  straight  rods; 
some  have  aerial  hyphae  branching  from  them  and  these  aerial  hyphae  terminate 
in  a  row  of  round,  irregular-sized  conidia;  club-shaped  forms  are  not  observed. 
Cultures  do  not  show  rosette  forms.  Involution  forms,  somewhat  similar  to, 
but  larger,  than  involution  forms  of  diphtheria  bacilli,  are  observed  in  very 
old  cultures. 

Growth. — Streptothrix  actinomyces  is  equally  adapted  to  aerobic  and  an- 
aerobic cultivation.  Growth  occurs  at  temperatures  between  2o°C.  and  5o°C., 
best  at  37°C. 


THE   HIGHER  BACTERIA  157 

Bouillon  shows  growth  after  5  or  6  days'  incubation  at  37°C.  A  whitish 
flocculent  sediment  and  a  whitish  surface  pellicle  forms  without  clouding  the 
medium. 

Glycerin  bouillon  cultures  have  the  same  appearance  as  plain  bouillon 
cultures. 

Gelatin. — Small,  round,  white,  glistening  colonies  appear  after  5  or  6  days. 
They  continue  to  increase  in  size  for  several  weeks  and  become  yellowish  in  the 
center.  Growth  is  scant  on  this  medium  and  it  is  liquefied  very  slowly. 

Glycerin  agar  shows  growth  in  2  or  3  days.  At  first  small,  wrinkled,  dry, 
white  colonies  appear;  these  coalesce  and  form  a  dry,  wrinkled  pellicle,  which 
may  be  white,  yellowish- white  or  yellow;  aerial  hyphae  appear  as  irregular 
projections  from  the  surface. 

Potato. — Growth  first  appears  similar  to  that  on  agar;  the  pellicle,  as  it 
ages,  is  first  yellow,  then  brown  and  finally  black. 

Milk  is  not  coagulated. 

Resistance. — Streptothrix  actinomyces  is  more  resistant  to  most  germicidal 
agents  than  the  tubercle  bacillus. 

Toxin. — Streptothrix  actinomyces  produces  an  intracellular  toxin. 

Pathogenesis. — Actinomycosis  is  most  frequently  seen  in  cattle,  the  site  of 
infection  in  most  cases  being  the  lower  jaw.  This  results  from  eating  grain  or 
grass  covered  with  actinomyces.  When  taken  into  the  mouth  an  abrasion  of 
the  mucosa  or  a  necrotic  tooth  offers  a  favorable  site  for  infection;  the  organism 
enters  and  the  disease  progresses.  Its  course  is  chronic  and  gradually  a  large, 
hard  swelling  forms,  a  condition  commonly  known  as  "lumpy  jaw;"  eventually 
suppuration  occurs,  then  spontaneous  evacuation.  Pus  oozes  from  the  opening. 

Infection  does  not  always  involve  the  jaw.  It  may  be  localized  in  the  tongue, 
skin,  peritoneum,  pleura  or  lungs  and  septicemia  or  generalized  infections  have 
been  observed. 

When  the  lungs,  pleura  or  peritoneum  are  involved  the  condition  simulates 
tuberculosis.  In  man  conditions  following  infection  are  similar  to  those  ob- 
served in  cattle.  The  disease  affects,  most  frequently,  those  engaged  in  agri- 
cultural occupations  and  the  care  of  cattle. 

Horses,  sheep,  and  hogs  are  sometimes  infected  and  the  disease  can  be  pro- 
duced in  rabbits  and  guinea-pigs  by  inoculation. 

Usually  secondary  infection  occurs;  staphylococci,  streptococci  and  other 
organisms  are  nearly  always  present  in  the  pus  discharged  from  actinomycotic 
lesions. 

In  the  sputum  from  pulmonary  cases  tubercle  bacilli  may  be  found,  together 
with  Streptothrix  actinomycosis,  and  should  be  sought  for  in  such  cases. 

Diagnosis. — Fluid  or  pus  from  suspected  cases  should  be  carefully  examined 
for  yellowish,  hard  granules.  If  any  are  found  they  should  be  crushed  between 
a  slide  and  cover  glass  and  examined  for  rosette-like  masses  of  club-shaped 
bodies  and  for  filaments.  If  granules  are  not  discovered,  then  the  pus  is  smeared 
on  a  slide  and  examined  for  rosettes.  The  suspected  material  must  be  examined 
as  soon  after  removal  from  the  lesion  as  possible. 


158  MEDICAL  BACTERIOLOGY 

The  finding  of  typical  rosettes,  clubs  and  filaments  establishes  the  diagnosis. 

When,  for  more  detailed  study,  cultures  are  desired  the  actinomyces  must  be 
isolated  from  other  organisms  present  in  the  pus.  The  technique  for  this  is  as 
follows : 

Lift  a  dozen  or  more  of  the  granules  from  the  pus  and  place  on  gelatin  plates  \ 
incubate  these  plates  at  37°C.  for  2  days;  most  of  the  granules  will  be  surrounded 
with  colonies  of  contaminating  bacteria;  search  for  granules  free  from  other 
bacteria  and  remove  these  with  a  sterile  loop  and  plant  on  slant  tubes  of  glycerin 
agar  and  tubes  of  LoefBer's  blood  serum;  incubate  these  tubes  at  37°C.  Many 
tubes  should  be  planted,  as  some  will  show  no  growth  and  others  will  contain 
organisms  other  than  actinomyces;  tubes  containing  only  strep  to  thrix  actino- 
myces will  show  growth  in  4  to  6  days. 

STREPTOTHRIX  MADURJE 

Streptothrix  madurae  is  found  in  the  lesions  and  pus  and  exudate  of  a  dis- 
ease known  as  "madura  foot." 

Morphology. — Granules  removed  from  lesions,  exudate  or  pus,  consist  of 
interlaced,  branching  filaments.  Organisms  removed  from  cultures  have  the 
same  appearance;  in  addition  they  show  spores  arranged  singly,  in  pairs,  and 
irregular  masses. 

Staining  readily  occurs  with  the  usual  anilin  dyes.     It  is  Gram  positive. 

Growth. — Streptothrix  madurae  is  an  obligate  aerobe  and  grows  best  at 
37°C.  In  bouillon,  gelatin,  agar,  egg  and  serum  growth  is  scant  when  it  takes 
place  at  all. 

Glucose  Glycerin  Agar. — After  several  days,  round,  smooth,  elevated, 
yellowish- white  colonies  appear;  as  they  age  they  increase  in  size,  become  pink 
and  later  red.  Umbilication  may  occur  or  the  colonies  may  coalesce. 

Potato. — ;Warty  colonies  develop  on  this  medium ;  at  first  white,  they  gradu- 
ally change  to  deep  red. 

Milk  is  not  coagulated. 

Hay  Infusion. — Flocculi  form  after  several  days;  some  precipitate  and  some 
float.  Those  exposed  to  the  oxygen  after  several  weeks  turn  from  white  to 
pink.  The  medium  is  not  clouded. 

Pathogenesis. — Streptothrix  madurae  causes  a  chronic  productive  inflam- 
matory condition  of  the  foot;  associated  nodular  swellings  give  a  warty  appear- 
ance to  the  surface.  After  a  time  suppuration  occurs,  followed  by  spontaneous 
rupture  and  sinus  formation.  The  exudate  or  pus  which  oozes  from  the  sinus 
contains  small  white,  yellow  or  black  granules  similar  to  those  found  in 
actinomycosis. 

Rarely  the  hand  is  affected.  The  disease  has  been  observed  in  man  only. 
Animals  are  immune. 

Diagnosis. — In  suspected  cases  the  pus  is  inspected  for  granules,  and  these 
are  stained  and  examined  microscopically. 

Cultures  are  obtained  by  asepticizing  the  skin  over  a  nodule,  making  an 
incision  and  removing  some  fluid  from  the  nodule  with  a  sterile  tube  and  trans- 
ferring it  to  hay  infusion  or  glycerin  glucose  agar. 


THE   HIGHER  BACTERIA  159 

ACHORION  SCHONLEINI 

Achorion  schonleini  is  present  in  the  hairs  and  scabs  of  tinea  favosa  or  favus. 

Morphology. — When  one  of  the  crusts  containing  a  hair  is  removed  from 
the  affected  area  and  examined  microscopically,  branched  chains  of  mycelial 
spores  are  observed  within  the  hair.  These  spores  are  round,  or  nearly  so,  and 
measure  from  3  to  6  M  in  diameter.  Here  and  there  along  the  hair  mycelial 
filaments  occur  and  in  the  crusts  or  epithelium  adjacent  to  them  masses  of 
tangled  filaments  occur.  The  filaments  are  delicate  and  vary  in  length;  some 
show  several  branches  and  others  do  not  branch. 

Growth. — Achorion  schonleini  is  aerobic  and  grows  at  temperatures  between 
I5°C.  and  38°C.,  best  at  33°C. 

Neutral  Glycerin  Bouillon. — A  dark  yellow  surface  pellicle  form  on  this 
medium. 

Neutral  Glycerin  Agar. — A  dark  yellow,  dry,  wrinkled,  umbilicated.  film 
develops. 

Potato. — Growth  first  is  grayish  yellow;  then  darker,  until  the  potato  turns 
brown.  It  covers  the  surface  with  a  thick  film. 

Pathogenesis. — Achorion  schonleini  is  the  cause  of  favus. 

"Tinea  favosa,  or  favus,  is  a  contagious  vegetable  parasitic  disease  of  the 
skin,  characterized  by  pin-head  to  pea-sized,  friable,  umbilicated,  cup-shaped, 
yellow  crusts,  each  usually  perforated  by  a  hair.  It  is  usually  met  with  upon 
the  scalp,  but  may  occur  upon  any  part  of  the  integument"  (Stelwagon). 

Achorion  schonleini,  or  a  closely  related  organism,  causes  favus  of  dogs  and 
mice. 

Diagnosis. — Bacteriological  diagnosis  is  based  on  microscopic  examination 
of  crusts  and  hairs  removed  from  affected  surfaces. 

The  crusts  and  hairs  are  placed  in  a  drop  of  20  to  40  per  cent,  potassium 
hydrate  solution  on  a  slide  and  a  cover  glass  is  drdpped  upon  it.  Gently  warm 
the  preparation  for  a  few  seconds  and  then  examine.  When  it  is  desirable  to 
keep  preparations  for  a  time,  after  warming  the  slide  is  placed  on  a  cold  surface 
to  rapidly  cool  it  and  when  cool  a  drop  of  eosin-tinted  glycerin  is  allowed  to  run 
between  cover  and  slide. 

TRICHOPHYTON  AND  MICROSPORON  AUDONINI 

Tinia  trichophytina,  or  ring  worm,  is  a  vegetable  parasitic  disease  which 
affects  various  portions  of  the  epithelial  surface  of  the  body,  chiefly  the  scalp 
or  bearded  region  of  the  trunk.  Several  triophy  ta  and  the  microsporon  audouini 
act  as  exciting  causes  of  the  disease. 

The  same  condition  has  been  observed  in  dogs  and  other  animals. 

Diagnosis. — Bacteriological  diagnosis  is  based  upon  microscopic  examina- 
tion of  hairs  removed  from  the  center  and  outer  zone  of  diseased  areas. 

The  hairs  are  placed  in  a  drop  of  20  to  40  per  cent,  potassium  hydrate  solu- 
tion and  a  cover  glass  dropped  on  them.  Several  slides  should  be  gently  warmed 
and  allowed  to  stand  at  room  temperature  for  several  minutes  before  examina- 
tion. Other  slides  should  be  heated  until  steam  arises  and  then  placed  on  a 


l6o  MEDICAL  BACTERIOLOGY 

cool  surface  to  rapidly  reduce  the  temperature,  then  examined.  The  micro- 
scopic appearance  of  the  most  common  forms  is  as  follows : 

Trichophyton  megalosporum  endothrix.  Affected  hairs  are  swollen  and 
rilled  with  spores  arranged  in  chains.  These  spores  are  5  to  6  M  in  diameter. 

Trichophyton  microides  ectothrix,  spores  no  larger  than  tricophyton  megalo- 
sporon  endothrix,  are  observed  both  within  and  surrounding  hairs. 

Trichophyton  megasporon  ectothrix,  spores  from  8  to  15  //,  in  diameter,  are 
found  both  within  and  surrounding  the  hairs. 

Microsporon  audouini,  affected  hairs  are  covered  with  irregularly  arranged 
masses  of  spores.  These  spores  are  round  and  measure  from  i  to  3  /j  in 
diameter.  They  never  penetrate  the  hair. 

MICROSPORON  FURFUR 

Microsporon  furfur  is  the  cause  of  tinea  versicolor  (pityriasis  versicolor),  a 
skin  disease  showing  a  yellow  macular  eruption  on  the  upper  part  of  the  trunk. 

Diagnosis. — Scrape  the  lesions  with  a  knife  or  glass,  place  the  scrapings  in 
a  drop  of  potassium  hydrate  solution  on  a  slide  and  cover  with  a  cover  glass. 

Collections  of  filaments  and  spores  are  observed  between  the  epithelial  cells. 

The  filaments  are  3  to  4  /*  in  diameter,  septate  and  branched. 

The  spores  occur  singly  and  in  irregular  masses;  they  are  spherical,  3  to  4  /tin 
diameter  and  are  encapsulated. 


CHAPTER  XLV 
HYPHOMYCETES  (Molds) 


Molds  are  very  widely  distributed  in  nature,  their  spores  are  so  nearly 
ubiquitous  that  any  article  containing  pabulum,  exposed  in  a  dark,  moist 
atmosphere,  is  soon  covered  with  a  growth. 

Only  a  few  molds  are  pathogenic  and  infection  with  such  organisms  is  com- 
paratively rare. 

From  time  to  time  various  hyphomycetes,  but  most  frequently  the  asper- 


FIG.  31. — MOLD  FUNGUS  DEVELOPING  AS  A  CONTAMINATION  OCCURRING  ON  AGAR. 

(8  eyepiece;  %  objective.) 

gillus,  are  reported  as  the  cause  of  pulmonary  infection  in  man,  simulating 
tuberculosis.  As  the  method  of  staining  sputum  to  disclose  tubercle  bacilli  is 
not  as  apt  to  reveal  hyphomycetes  as  Gram's  method,  it  is  probably  the  best 
practice  to  stain  specimens  of  sputum  for  diagnosis  by  both  methods  and  in  some 
cases  at  least  to  make  cultures.  Sputum  culture  by  the  method  of  Petroff  will 
disclose  hyphomycetes  quite  as  well  as  tubercle  bacilli. 

Though  pathogenic  molds  will  grow  on  most  of  the  ordinary  culture  media, 
Sabouraud's  is  best:  Maltose,  40  Gm.;  Peptone,  10  Gm.;  Agar,  15  Gm.; 
Water,  1000  c.c. 

Adjust  reaction  to   +  2  and  autoclave. 

Growth  usually  does  not  appear  until  after  4  or  5  days'  incubation,  frequently 
longer. 

The  hyphae,  mycelia  and  spores  of  molds  are  of  such  size  that  their  struc- 
11  161 


162 


MEDICAL  BACTERIOLOGY 


ture  and  arrangement  is  discernible  when  examined  unstained  with  a  low-power 

lens  (%  to  ^6  objective). 

Molds,  hyphomycetes  or  eumycetes  are  all  alike  and  differ  from  the  algae 

in  that  they  (the  molds)  do  not  possess  chlorophyl. 

Pathogenic  hyphomycetes  belong  to  one  of  three  classes: 

First.    Fungi     Imperfect! — To    this    group   is   relegated   all   forms   not 

thoroughly  studied  and  those  which  do  not  belong  to  either  of  the  other  two 

classes.     Fungi  imperfecti  includes  most  of  the  pathogenic  molds. 

MOLDS,  HYPHOMYCETES  OR  EUMYCETES 

Phycomycetes  Mycomycetes 

Hyphae  or  mycelia  show  no  partitions,  en-     Hyphae  or  Mycelia  show  partitions, 
tire  meshwork  of  a  single  organism  consist- 
ing of  one  multinucleated  cell. 

Reproduction  sexual  and  asexual,  no  defi-  Reproduction  usually  asexual,  only  definite 

nite  basidium  or  ascus.  basidium  or  ascus. 


Mucor 

Mucor  Mucedo 
Mucor  Pusillus 
Mucor  Corymbifer 


Aspergillus 

A.  Subflavus 
A.  Fumigatus 
A.  Flavus 
A.  Niger 
A.  Concentricus 
A.  Albus 


Penicillium 

P.  Galucum 
P.  Crustaceum 


FIG.  32. — MUCOR  CORYMBIFER.     (From  Plant  after  Lichtheim.) 

Asperigillus  fumigatus  has  been  found  in  inflammatory  conditions  of  the 
upper  air  passages  and  in  the  sputum  of  persons  suffering  with  pulmonary  dis- 
ease simulating  tuberculosis. 


HYPHOMYCETES  163 

Aspergillus  fumigatus  produces  a  tubercular-like  infection  in  pigeons,  fowl, 
guinea-pigs  and  rabbits.  Growth  on  culture  media  is  at  first  whitish,  then  green 
or  greenish-blue;  later  it  becomes  brown  or  black. 

Penicillium  glaucum  has  been  found  in  chronic  inflammatory  conditions  of 
the  naso-pharynx  and  Eustachian  tube.  Growth  on  culture  media  is  green. 
This  mold  is  used  in  the  manufacture  of  certain  kinds  of  cheese. 

Mucor  mucedo  is  the  mold  most  commonly  observed  on  foodstuffs;  it  is 
whitish  and  appears  like  cotton.  This  organism  has  been  reported  as  the  excit- 
ing cause  in  several  cases  of  tubercular-like  infections  of  the  lungs,  in  which 
cases  it  was  found  in  the  sputum. 

The  development  of  hyphae,  basidia  and  sporulation  of  many  of  the  higher 
forms  of  bacteria,  molds  and  yeasts  can  be  studied  best  as  follows: 

Liquefy  a  tube  of  sterile  glucose  agar,  spread  a  small  drop  of  it  in  a  thin  even 
film  on  a  clean  slide  with  a  sterile  pipette,  and  when  solid  lightly  stroke  the 
surface  with  a  platinum  loop  charged  with  the  material  to  be  studied.  Place 
a  cover  glass  in  it  and  incubate  at  37°C.  At  frequent  intervals  place  on  micro- 
scope and  observe  growth  with  %  and  %  objectives  and  high-power  eyepiece 
i ox  to  i8x. 

Resistance. — The  spores  of  molds  are  frequently  quite  as  resistant  or  more 
resistant  to  the  germicidal  influence  of  heat,  light  and  chemicals  than  the  spores 
of  bacteria.  It  is  a  matter  of  practical  importance  to  know  that  formaldehyde 
is  less  germicidal  to  molds  than  to  bacterial  spores;  that  molds  are  especially 
susceptible  to  phenol  solutions,  and  very  weak  solutions  of  copper  sulphate, 
i :  1000  or  less,  will  prevent  their  development. 


CHAPTER  XL VI 

SACCHAROMYCETES 

(BLASTOMYCETES) 

Yeasts  are  widely  distributed  in  nature,  having  much  the  same  occurrence 
as  molds.  They  are  the  common  cause  of  fermentation  and  with  the  exception 
of  a  few  of  the  pathogenic  species  produce  an  endo-enzyme  that  splits  sugars 
into  alcohol  and  CO2.  In  addition  many  of  them  produce  other  ferments  that 
reduce  starches  and  other  complex  substances. 

Torula  or  wild  yeasts  are  chiefly  of  interest  as  frequent  and  annoying 
contaminants. 

The  cultivated  varieties  play  an  important  role  in  various  industries,  espe- 
cially wine-making,  brewing  and  baking. 

As  is  the  case  with  bacteria  and  hyphomycetes 
only  a  few  yeasts  are  pathogenic. 

Morphology. — Saccharomycetes  as  commonly  ob- 
served are  round  or  oval  unicellular  organisms, 
presenting  a  distinct  cell  wall — some  species  having 
a  double,  concentric  wall.  The  protoplasm  generally 
contains  numerous  small  structureless  granules,  and, 
less  frequently,  one  or  several  vacuoles. 

They  occur  singly,  in  chains  and  irregular  masses. 
As  a  whole,   individual   cells  are  sufficiently  larger 
than  cocci  and  the  spores  of  molds  to  be  distinguish- 
able from  them  on  sight,  but  exceptions  to  this  occur: 
FIG.  33-— YEAST  CELL. 

(Marshall.')  some  are  as  little  as  o.$fj.  in  diameter,  they  average 

10  ju  to  49  M  in  diameter. 

Different  species  differ  in  size  but  individual  differences  in  size  in  various 
cultures  of  a  single  species  under  different  conditions  is  great. 

Saccharomycetes  are  non-motile  and  quite  distinctly  discernible  when  not 
stained.  They  stain  readily  with  the  usual  dyes  and  are  Gram  positive. 

Growth. — There  are  both  aerobic  and  anaerobic  species.  Isolated  patho- 
genic species  are  aerobic  and  grow  well  in  all  the  ordinary  culture  media.  Incu- 
bated at  37°C.,  growth  becomes  apparent  in  from  24  hours  to  5  weeks  and  con- 
tinues for  a  long  time. 

Young  colonies  on  agar  are  round,  dry,  smooth,  waxy,  white  or  yellowish, 
firm  and  adherent  and  may  attain  a  diameter  of  i  centimeter.  Later  they 
become  wrinkled,  moist  and  develop  aerial  hyphae. 

Reported  studies  of  pathogenic  species  of  saccharomyces  indicate  that  many, 
if  not  all,  under  certain  conditions  form  mycelia  and  aerial  hyphae. 

164 


SACCHAROMYETES 


165 


In  tissue  and  actively  growing  young  cultures,  all  species  reproduce  by 
budding,  numerous  cells  showing  one  or  more  buds.  These  buds,  which  first 
appear  as  small  globular  protrusions  from  the  cell  wall,  rapidly  increase  in  size 
and  eventually  split  off,  becoming  mature  cells. 

Some,  if  not  all  species,  under  certain  conditions,  reproduce  by  sporulation 
and  present  cells  containing  one  or  more  spherical  spores — usually  four. 

Pathogenesis.— Though  comparatively  rare,  infection  in  man  with  saccharo- 
mycetes  is  much  more  frequent  than  infection  with  molds.  The  species  in- 
fecting man  is  also  pathogenic  for  dogs,  cats  and  mice,  especially  the  latter. 
The  source  of  infection  is  obscure,  the  onset  insidious,  the  disease  essentially 
chronic  and  frequently  fatal.  Reported  cases  tend  to  indicate  that  the  nature 
of  such  infections  usually  goes  unrecognized. 


FIG.  34. — YEAST  CELLS  STAINED  WITH  FUCHSIN.     (  Xiooo.)     (MacNeal.) 

At  least  three  species  are  pathogenic  for  man  (the  investigations  of  Rabino- 
witsch  indicate  more) : 

Oidium  Albicans  (Endomyces  albicans),  which  attacks  the  buccal  mucous 
membrane  of  ill-cared-for,  debilitated  infants  and,  less  often,  adults.  It  forms 
a  white  growth  or  pseudomembrane  on  the  affected  part  and  in  some  cases  may 
cause  necrosis  of  underlying  tissue.  The  condition  is  known  as  Oidiomycosis, 
thrush  or  babies'  sore  mouth. 

Zymonema  Gilchristi,  which  attacks  the  skin,  producing  a  condition  referred 
to  as  Blastomycetic  Dermatitis. 

Sac  char  omyces  Tumefacians,  which  may  be  a  localized  infection  causing  a 
tumor-like  mass,  or  extensive  ulceration,  of  any  part  of  the  body,  or  a  localized 
pulmonary  infection  simulating  pulmonary  tuberculosis  or  a  generalized  infec- 
tion with  multiple  abscess  formation. 

Diagnosis. — Microscopic  examination  of  smear  made  from  the  affected  mem- 


1 66  MEDICAL  BACTERIOLOGY 

brane  in  oidiomycosis,  or  made  from  the  serum  or  pus  expressed  from  skin 
lesions  in  bias tomyce tic  dermatitis,  or  from  the  sputum  in  pulmonary  blasto- 
mycosis,  or  pus  of  abscesses,  readily  discloses  the  nature  of  the  infection,  the 
large,  round  and  oval,  budding  cells  with  a  distinct  wall  being  characteristic. 

Sections  of  involved  tissue  present  numerous  identical  cells. 

Saccharomyces  Neoformans,  a  yeast  frequently  found  in  fermented  fruit  and 
advanced  as  the  cause  of  cancer  by  Sanfelice,  probably  is  not  pathogenic  and 
has  been  rejected  as  the  cause  of  carcinoma. 

COCCIDIOIDES  IMMITIS 

Coccidioides  Immitis  is  the  cause  of  a  disease  in  man — Coccidiosis — which 
begins  as  a  papular  eruption,  the  papules-  later  coalescing,  become  pustules, 
which  in  turn  are  followed  by  ulceration  and  a  purulent  discharge. 

The  infection  may  remain  localized  in  the  skin  for  months,  being  confined 
to  the  integument  of  one  extremity  or  involving  a  larger  area.  Eventually  in 
most  cases  the  lymphatic  glands  are  attacked  and  general  disseminated  infec- 
tion with  clinical  signs  and  pathological  changes  resembling  miliary  tubercu- 
losis mark  the  last  stage  of  its  fatal  course. 

Morphology. — Coccidioides  Immitis,  in  the  purulent  discharge  from  lesions, 
in  the  giant  cells  of  tubercles  found  in  affected  tissue  and  in  material  taken  from 
buboes,  appears  as  round,  thick- walled,  yeast-like  cells,  20  /z  to  40  /i  in  diameter, 
some  of  which  contain  spores. 

In  spreads  made  from  cultures  organisms  identical  to  those  found. in  tissue 
are  observed  and  in  addition  some  show  budding  and  mycelia. 

Growth. — Occurs  aerobically  at  room  and  body  temperature  on  agar, 
glycerin  agar  and  glucose  agar,  neutral  or  acid  in  reaction. 

Pathogenesis. — Subcutaneous  inoculation  of  pus  or  fluid  from  lesions  and 
of  cultures  produces  disease  in  guinea-pigs,  rabbits  and  monkeys  similar  to 
that  observed  in  man. 

Diagnosis. — Make  smears  for  microscopic  examination  and  cultures  on 
Sabouraud's  agar  of  purulent  discharge  from  skin  lesion,  or,  if  the  disease  has 
not  progressed  beyond  the  papular  stage,  excise  a  papule,  fix,  section,  stain  and 
examine  serial  sections. 

When  buboes  exist,  if  organisms  have  not  been  found  in  superficial  lesions, 
massage  an  enlarged  gland,  aspirate  as  much  as  possible  of  its  contents  with  a 
syringe  and  needle  and  examine  the  same  as  pus. 

For  a  detailed  description  of  systemic  blastomycosis  see  Stober,  A.  M., 
Archives  Int.  Med.,  xiii,  No.  4,  April,  1914. 


CHAPTER  XLVII 
MONILA 

Monila  resemble  Oidium  (endomyces)  albicans  and  are  closely  related. 
When  first  described  as  a  separate  genus  the  distinguishing  characteristic  of 
monila  was  stated  to  be  the  absence  of  ascosporulation. 

Based  on  variations  in  effect  on  litmus  milk,  gelatin,  the  carbohydrates  and 
agglutination  reactions,  many  species  have  been  recognized  in  recent  years. 
Castellani  has  isolated  and  described  the  following:  monila  intestinalis,  monila 
faecalis,  monila  insolita  and  monila  tropicalis — all  obtained  from  the  saliva, 
scrapings  from  the  tongue  or  feces  of  patients  afflicted  with  sprue — of  which 
disease  he  does  not  believe  they  are  the  cause.  Bahr  also  has  recovered  an 
organism,  described  as  monila  albicans,  from  the  tongue  and  feces  of  sprue 
patients  and  he  believes  it  the  cause  of  their  disease. 

In  scrapings  from  the  tongue,  in  the  saliva  and  feces  of  patients  having  sprue, 
Ashford  has  regularly  found  a  monila  that  in  its  parasitic  state  has  the  same 
morphology  as  oidium  albicans,  that  produces  mycelia  but  does  not  reproduce 
by  ascospores  in  its  vegetative  state,  that  forms  gas  and  acid  in  glucose,  levulose 
and  maltose  and  does  not  form  gas  but  acidulates  saccharose  and  galactose 
media;  it  does  not  liquefy  gelatin  or  blood  serum  and  does  not  coagulate  milk  but 
makes  it  alkaline.  This  organism  is  pathogenic  for  rabbits  and  is  believed  by 
Ashford  to  be  the  cause  of  sprue.  He  believes  the  infection  may  be  conveyed 
to  man  by  bread  and  states  in  reference  to  primary  diagnostic  cultures,  "I 
consider  this  medium  as  specific  for  monila  as  Loeffler's  blood  serum  mixture 
is  for  the  bacillus  diphtheriae" — meaning  Sabouraud's  glucose  agar  (4  per  cent, 
glucose)  having  a  reaction  of  -f-2. 

A  reported  case  of  pulmonary  moniliasis  has  been  observed  in  the  Johns 
Hopkins  Hospital. 


167 


CHAPTER  XLVIII 
SPOROTRICHUM  SCHENKII 

Sporotrichum  Schenkii  occurs  in  the  lesions  of  sporotrichosis,  a  chronic 
infection  marked  by  the  occurrence  of  multiple  subcutaneous  gumma-like  masses 
which  liquefy  and  discharge  material  similar  to  pus.  Occasionally  other  parts 
are  affected,  infections  of  the  buccal  and  pharyngeal  mucous  membrane,  lym- 
phatic glands,  bones  and  synovial  membrane  having  fceen  reported. 

Morphology. — Fluid  and  sections  of  tissue  from  lesions  show  elongated  oval, 
yeast-like  cells  3  ju  to  6  ju  long.  Cultures  show  long  delicate,  septate,  branched 
filaments  with  clusters  of  from  2  to  12  or  more  brown  spherical  and  oval  spores, 
about  2  fj,  in  diameter  at  their  ends. 

Culture  growth  appears  on  Sabourand's  agar,  incubated  aerobically  at  room 
temperature,  in  5  to  10  days.  At  first  white,  as  it  ages  the  growth  darkens, 
spreads  over  the  entire  surface  and  becomes  brown. 

Glucose  Broth. — After  a  number  of  days  a  white  pellicle  forms  on  the  sur- 
face, the  medium  remaining  clear.  In  time  the  pellicle  precipitates  and  a  second 
surface  pellicle  may  form. 

Diagnosis. — Massage  hard  gumma-like  masses  or  glands,  if  soft  ones  are  not 
present.  With  a  sterile  syringe  and  large  bone  needle  withdraw  the  contents, 
plant  as  much  as  possible  up  to  i  cc.  on  Sabouraud's  agar  slants  and  spread  on 
slides  for  immediate  microscopic  examination.  Examine  both  unstained  and 
stained  preparations. 

When  fluid  cannot  be  obtained  or  contains  no  organisms,  pieces  of  diseased 
tissue  should  be  excised,  cultured,  and  also  sectioned  for  microscopic  examination. 


1 68 


CHAPTER  XLIX 
INFECTIOUS  DISEASES  OF  UNKNOWN  CAUSATION 

Of  the  diseases  afflicting  man  that  have  been  differentiated  about  one-half 
are  caused  by  known  organisms. 

The  method  of  transmission  from  person  to  person,  the  mode  of  onset  and 
the  course  of  many  other  diseases  indicate  that  they  too  are  due  to  infection, 
although  attempts  to  isolate  and  identify  the  causative  organisms  have  failed. 
The  success  of  research  from  time  to  time  is  happily  reducing  this  group.  It 
is  yet  too  early  for  the  medical  profession  to  have  adequately  tested  and  con- 
firmed the  remarkable  discoveries  of  the  last  4  years  relative  to  typhus  fever,  but 
the  evidence  strongly  suggests  that  the  specific  cause  of  this  disease  has  been 
disclosed. 

Bacillus  typhi-exanthematici,  first  described  by  Plotz,  Olitsky  and  Baehr, 
has  been  obtained  in  blood  cultures  from  typhus  patients,  using  a  2  per  cent, 
glucose-ascitic-fluid-agar  medium.  It  has  also  been  isolated  from  lice  caught 
where  the  disease  prevailed  and  from  mice,  guinea-pigs  and  monkeys  injected 
with  the  blood  of  typhus  patients. 

It  is  a  Gram-positive,  obligate  anaerobic  bacillus.  The  spleen  of  mice 
inoculated  with  this  organism  is  said  to  show  characteristic  lesions. 

Infection  of  man  has  been  known  for  a  long  time  to  depend  upon  transmis- 
sion by  the  body  louse  and  hence  the  prevention  of  typhus  epidemics  and  the 
curtailment  of  existing  epidemics  is  effected  by  extermination  of  body  lice. 

Occasionally  before  crisis,  frequently  at  the  time  of  the  crisis,  and  in  nearly 
all  cases  after  crisis,  the  blood  serum  of  typhus  patients  will  cause  agglutination 
of  the  bacillus  typhi-exanthematici,  and  will  cause  complement  fixation  with 
this  antigen. 

Opinion  differs  widely  as  to  the  prophylactic  and  therapeutic  value  of  vac- 
cine. It  is  worthy  of  note  that  Strong  has  not  yet  indorsed  the  conclusions  of 
those  who  believe  in  the  efficacy  of  this  vaccine. 

ACUTE  ANTERIOR  POLIOMYELITIS 

(Epidemic  Poliomyelitis) 

Acute  anterior  poliomyelitis  is  a  disease  caused  by  a  filterable  virus.  Emul- 
sions of  the  brain  and  cord  of  persons  dead  of  the  disease  injected  into  monkeys 
and  rabbits  frequently  produce  malaise,  fever,  paralysis  and  death. 

One  attack  confers  immunity  and  when  inoculation  is  not  fatal  immunity 
results. 

Flexner  and  Noguchi  isolated  a  minute  coccus  and  cultivated  it  anaerobic- 
ally  in  fluid  serum.  They  believe  this  organism  the  specific  cause  of  the  disease 
but  their  view  has  not  been  generally  accepted. 

169 


1 70  MEDICAL  BACTERIOLOGY 

Dixon  and  Rucker  also  succeeded  in  isolating  a  similar  but  probably  not 
identical  organism  from  the  brain  and  cord  of  patients  dead  of  the  disease. 
At  first  they  were  inclined  to  believe  it  the  causative  organism  but  later  rejected 
it  as  the  specific  etiological  factor.  Recently  Rosenow  and  others  have  obtained 
cultures  of  an  organism -described  as  pleomorphic  streptococcus  from  the  nasal 
mucosa,  the  pharynx  and  tonsils  of  poliomyelitis  patients.  They  have  isolated 
the  same  organism  from  the  ventricular  fluid,  brain  and  cord  of  fatal  cases. 

The  pleomorphic  streptococcus  is  described  as  a  coccus  arranged  in  pairs 
like  pneumococci  (but  without  a  capsule)  and  in  chains.  Marked  variations  in 
size  are  observed.  In  dextrose-ascitic  broth  growth  occurs  under  aerobic  and 
anaerobic  conditions  but  is  more  rapid  in  an  aerobic  atmosphere. 

Incubated  at  body  temperature  growth  appears  in  from  3  to  5  days  and  con- 
tinues for  several  weeks.  Old  cultures  show  large  and  club-shaped  forms. 
Young  cultures  are  Gram  positive  and  old  cultures  Gram  negative. 

Growth  occurs  on  blood-agar  incubated  at  body  temperature,  aerobically. 
Some  describe  the  colonies  on  this  medium  as  small,  white,  and  dry,  without 
any  change  in  the  appearance  of  the  underlying  or  surrounding  medium;  others 
describe  a  slight  clear  zone  or  greenish  zone  surrounding  each  colony. 

Various  observers  have  reported  inoculation  of  rabbits  by  placing  pleo- 
morphic streptococcus  upon  the  nasal  mucosa,  by  injecting  it  subcutaneously, 
by  injecting  it  into  a  nerve  or  beneath  the  dura.  In  such  cases  symptoms  and 
pathological  changes  associated  with  the  pleomorphic  streptococcus  and  re- 
covery of  the  organism  have  convinced  Rosenow  and  others  that  it  is  the  specific 
cause  of  poliomyelitis  but  at  present  we  must  consider  their  case  not  proven. 

Netter  has  discovered  that  the  blood  serum  of  persons  recovered  from  polio- 
myelitis possesses  distinct  therapeutic  value,  practically  undiminished  through- 
out their  life.  He  recommends  the  intraspinal  injection  of  such  serum  in  the 
treatment  of  the  disease,  from  5  to  13  cc.  each  day  for  i  week. 

SPIROCHJETA  ICTEROHEMORRHAGA 
(Spirochaeta  Nodosa) 

The  discoveries  recently  reported  by  Ito,  Matsuzaki,  Uhlenhuth  and  Fromme 
seem  to  indicate  that  Weil's  disease  is  caused  by  infection  and  that  the  offending 
organism  is  a  small  spirochete. 

Ito  and  Matsuzaki  have  described  three  closely  allied  strains  of  spirochaeta 
icterohemorrhaga  that  apparently  are  the  same  as  spirochaeta  nodosa. 

This  organism  (or  organisms)  has  been  found  constantly  associated  with 
Weil's  disease.  Blood  cultures,  especially  when  made  on  the  fourth  or  fifth 
day  of  the  disease  are  usually  positive.  The  organism  is  occasionally  present 
in  the  sputum,  urine,  or  feces  of  patients  during  the  disease  and  sometimes 
as  long  as  50  days  after  recovery. 

Inoculation  of  mice,  rats  and  guinea-pigs  with  cultures  obtained  from 
patients  causes  disease  the  same  as  in  man  and  identical  to  the  disturbance  pro- 
duced by  inoculation  of  these  animals  with  the  urine  of  patients. 


INFECTIOUS   DISEASES   OF   UNKNOWN  CAUSATION  171 

Virulent  spirochaeta  are  also  present  in  the  urine  and  feces  of  inoculated 
animals. 

Spirochaeta  icterohemorrhaga  is  anaerobic  and  facultative  aerobic.  Uhlen- 
huth  and  Fromme  cultivate  it  in  tubes  of  rabbit  serum  diluted  with  normal  salt 
solution  and  cover  this  medium  with.a  layer  of  oil  to  exclude  air. 

The  serum  of  persons  and  animals,  after  recovery  from  Weil's  disease,  con- 
'tains  antibodies  which  will  produce  Pfeiffer's  phenomenon  when  brought  into 
contact  with  spirochaeta  icterohemorrhaga. 

GANGRENE 

The  type  of  gangrene  due  to  infection  has  been  attributed  to  several  bacilli 
that  were  looked  upon  as  most  constantly  associated  with  the  disease  and,  there- 
fore, probably  a  specific  factor. 

It  is  now  well  established  that  gangrene  due  to  infection  may  be  produced  by 
a  variety  of  organisms,  the  majority  of  which  are  obligate  anaerobic. 

Small  pox,  scarlet  fever,  yellow  fever  and  measles  are  all  infectious  dis- 
eases caused  by  unknown  microorganisms. 

Small  Pox. — The  virus  of  this  disease  is  present  in  the  pustules  that  develop 
on  the  skin  of  infected  persons.  One  attack  of  the  disease  confers  immunity. 
Cow  pox  is  a  disease  of  cattle  similar  to  small  pox  in  man,  and  the  virus  of  cow 
pox  is  present  in  the  pustules  which  develop  on  the  epithelial  surface  of  infected 
animals.  In  1798  Jenner  discovered  that  the  virus  taken  from  the  pustules  of 
cow  pox  and  applied  to  the  skin  of  human  beings  would  inoculate  them  pro- 
ducing a  mild  infection  which  immunized  them  against  small  pox. 

Scarlet  Fever. — Although  streptococci  are  constantly  associated  with  this 
disease,  the  majority  of  the  medical  profession  does  not  believe  that  they  are 
the  specific  etiological  factor.  The  virus  of  scarlet  fever  is  present  in  the  blood 
in  the  early  days  of  the  disease  and  is  believed  by  many  to  also  be  present  in  the 
desquamated  epithelium. 

Yellow  Fever. — The  virus  of  this  disease  is  present  in  the  blood  of  affected 
persons  as  has  been  proven  by  withdrawing  such  blood  and  injecting  it  into 
healthy  persons,  thereby  inoculating  them.  This  virus  is  never  transferred 
directly  from  man  to  man.  Man  is  always  infected  through  the  bite  of  a  mos- 
quito. The  mosquito  in  turn  acquires  the  organism  by  feeding  on  the  blood  of 
yellow  fever  patients. 

Measles. — As  shown  by  Anderson  the  virus  measles  is  present  in  the  nasal 
secretions  and  the  circulating  blood  prior  to  the  development  of  the  rash. 

Anderson's  work  has  clearly  shown  that  this  virus  is  transmitted  from  man 
to  man  by  direct  intimate  contact  during  the  period  of  coryza  and  before  the 
rash  develops. 

There  is  much  evidence  to  indicate  that  scarlet  fever  is  probably  transmitted 
in  a  similar  manner. 


BACTERIOLOGY 
PART  II 

CHAPTER  I 
EXAMINATION  OF  WATER 

Water  Which  may  be  considered  potable,  without  suspicion,  contains  few 
or  no  bacteria  and  no  pathogenic  bacteria  (no  colon  bacilli).  Most  water  con- 
tains some  bacteria.  That  which  shows  100  colonies  per  cubic  centimeter  or 
less,  none  of  which  are  pathogenic,  should  be  considered  safe.  When  500 
colonies  per  cubic  centimeter  or  more  are  present,  even  though  none  of  them  are 
pathogenic,  it  must  be  considered  with  suspicion,  because  where  so  much  bac- 
terial life  can  be  found  in  water,  as  a  rule,  conditions  are  favorable  for  the 
entrance  of  disease-producing  germs  at  any  time. 

Colon  bacilli  in  water  are  not  always  of  human  origin;  in  populated  districts 
they  usually  are  and  hence  we  are  compelled  to  consider  the  presence  of  colon 
bacilli  in  water  evidence  of  dangerous  contamination. 

Colon  bacilli  in  water,  of  themselves,  probably  do  not  constitute  a  grave 
danger,  but  experience  has  shown  that,  as  a  rule,  colon  bacilli  in  water  indicates 
pollution  with  human  sewage,  and  such  pollution  eventually  results  in  an 
epidemic  of  typhoid  fever,  cholera  or  other  water-borne  diseases. 

It  is  always  difficult,  frequently  impossible,  to  detect  and  isolate  typhoid 
bacilli  and  other  pathogenic  bacteria  from  water,  even  when  such  organisms  are 
known  to  be  in  it.  The  colon  bacillus  can  be  detected  and  isolated  with  com- 
parative ease;  hence,  water  is  examined  for  colon  bacilli  and  judged  as  good  or 
bad,  according  to  the  presence  or  absence  of  the  colon  bacillus. 

Efficient  chemical  treatment  or  nitration  not  only  removes  or  destroys  all 
pathogenic  bacteria  and  colon  bacilli,  it  removes  or  destroys  more  than  95  per 
cent,  of  the  total  bacterial  content.  Hence,  in  estimating  the  efficiency  of 
chemical  treatment  or  nitration,  samples  are  obtained  before  and  after  treat- 
ment and  examined  to  determine  the  number  of  bacteria  per  cubic  centimeter, 
also  for  colon  bacilli. 

To  get  a  fair  sample  for  examination,  when  water  is  obtained  from  a  tap  or 
faucet,  the  water  should  be  allowed  to  flow  for  at  least  5  minutes  before  col- 
lecting a  specimen. 

If  it  is  to  be  obtained  from  a  spring,  lake  or  stream  the  person  collecting 
should  wash  his  hands  and  then  submerge  the  container  before  opening  it.  The 
container  should  be  kept  submerged  until  it  is  full  and  has  been  closed  or 
stoppered. 

173 


174  MEDICAL  BACTERIOLOGY 

When  a  sample  has  been  obtained  it  should  be  examined  as  soon  as  possible. 
If  time  must  elapse  before  examination,  the  sample  should  be  cooled  to  i5°C. 
and  maintained  at  or  below  that  temperature  until  examined. 

Samples  for  bacteriological  examination  are  collected  in  sterile  receptacles, 
measured  with  sterile  pipettes  and  cultured  in  sterilized  tubes  and  dishes. 

TECHNIQUE 

Fermentation  Test. — Shake  container  to  evenly  distribute  bacteria  that 
may  be  in  the  water.  Take  a  number  of  fermentation  tubes  containing  lactose 
broth  medium. 

Put    o.i  cc.  into  each  of  the  first  two  tubes. 

Put    0.2  cc.  into  each  of  the  second  two  tubes. 

Put    0.4  cc.  into  each  of  the  third  two  tubes. 

Put    0.8  cc.  into  each  of  the  fourth  two  tubes. 

Put    1.5  cc.  into  each  of  the  fifth  two  tubes. 

Put    3 .o  cc.  into  each  of  the  sixth  two  tubes. 

Put    6.0  cc.  into  each  of  the  seventh  two  tubes. 

Put  10. o  cc.  into  each  of  the  eighth  two  tubes 

Place  one  set  of  tubes  containing  the  various  amounts  of  water  in  an  incu- 
bator at  37°C.  and  the  others  in  an  incubator  at  room  temperature. 

Examine  these  tubes  at  24-hour  intervals  for  3  days. 

If  fermentation  occurs  gas  accumulates  at  the  top  of  the  tube.  When  fer- 
mentation occurs  it  is  due  to  the  presence  of  the  colon  bacillus  in  the  majority 
of  cases,  but  one  must  remember  that  other  organisms  can,  and  occasionally  do, 
cause  fermentation  even  when  the  water  is  free  of  colon  bacilli,  usually  bacillus 
Welchii. 

Plating. — A  number  of  tubes  of  litmus  lactose  agar  are  liquefied  by  placing 
in  a  water  bath  and  hearing  to  the  boiling  point.  They  are  then  cooled  to  45°C. 

Put  o.i  cc.  of  water  into  the  first  tube  and  empty  tube  into  Petri  dish. 
Put  o.  2  cc.  of  water  into  the  second  tube  and  empty  tube  into  Petri  dish. 
Put  0.4  cc.  of  water  into  the  third  tube  and  empty  tube  into  Petri  dish. 
Put  o .  8  cc.  of  water  into  the  fourth  tube  and  empty  tube  into  Petri  dish. 
Put  1.5  cc.  of  water  into  the  fifth  tube  and  empty  tube  into  Petri  dish. 
Put  2.0  cc.  of  water  into  the  sixth  tube  and  empty  tube  into  Petri  dish. 

Make  a  duplicate  set  of  plates,  using  2  or  3  per  cent,  plain  agar  having  a 
reaction  of  +1.5.  Incubate  one  set  at  room  temperature,  the  other  at  37°C. 

Count  the  colonies  every  24  hours  for  3  days. 

When  water  is  dropped  into  a  tube  of  agar  the  tube  should  be  inverted  several 
times  to  evenly  distribute  the  water  before  pouring  into  a  Petri  dish.  Care 
should  be  taken  to  distribute  the  liquid  agar  evenly  over  the  entire  bottom  of  the 
Petri  dish  and  the  dish  should  not  thereafter  be  tilted,  moved  nor  placed  in 
incubator  until  the  agar  has  solidified. 

The  colon  bacillus  and  other  organisms  that  ferment  lactose  turn  litmus  lac- 
tose agar  red.  When  red  colonies  appear  they  are  removed  from  the  plates 
with  a  sterile  platinum  loop  and  planted  in  Dunham's  solution,  litmus  milk, 


EXAMINATION    OF   WATER  175 

and  on  plain  agar  and  gelatin.  The  Dunham's  solution  is  tested  for  indol  and 
the  morphology,  motility  and  reaction  with  Gram's  staining  observed.  If 
necessary  further  differential  cultures  may  be  made. 

The  above  technique  should  be  employed  when  examining  a  new  or  unknown 
sample  of  water.  Samples  of  water  obtained  from  the  same  source  at  frequent 
intervals,  as  in  routine  work,  number  of  examinations  showing  that  the  bacteria 
contained  does  not  vary  to  any  great  extent,  do  not  need  to  be  planted  in  so 
many  different  quantities.  With  water  which  is  usually  potable,  it  is  sufficient 
to  plant  just  three  different  quantities  in  fermentation  tubes — 3  cc.,  5  cc.  and 
10  cc. — and  three  different  quantities  in  plates — i  cc.,  ij^  cc.,  and  2  cc. 


FlG.    35. — -WOLFHUGEL'S    COLONY    COUNTER. 

Water  which  is  grossly  contaminated  with  sewage  is  frequently  as  rich  in 
bacteria  as  dirty  milk  and  hence  must  be  diluted  with  sterile  distilled  water 
before  plating  or  making  fermentation  tubes. 

For  reasons  discussed  on  page  181  it  is  essential  in  making  a  numerical  count 
to  obtain  plates  that  show  between  20  and  200  colonies.  When  making  the 
count  all  colonies  that  are  visible  when  examined  through  a  reading  lens  that 
magnifies  three  diameters  are  to  be  counted. 

When  water  is  suspected  to  contain  the  cholera  spirillum,  to  100  cc.  of  the 
suspected  water  add  i  Gm.  peptone  (Witte's)  and  J^  Gm.  sodium  chloride, 
shake  to  mix  and  incubate  at  37°C.  in  a  tall,  narrow  container.  After  6  hours 
carefully  remove  several  loopsf  ul  from  the  top  and  transplant  as  when  examining 
feces  (pages  116  and  117). 


CHAPTER  II 
EXAMINATION  OF  MILK 

Milk  is  exceptionally  liable  to  contamination  with  bacteria  and  is  a  most 
favorable  medium  for  their  development.  Consequently  practically  all  of  it 
has  an  abundant  bacterial  content. 

Milk  that  shows  20,000  colonies  per  cubic  centimeter  or  less  is  extraordinarily 
clean.  Milk  that  shows  10,000  colonies  per  cubic  centimeter  or  less  is  seldom 
observed. 

In  winter,  average  city  milk  shows  from  5000  to  500,000  colonies  per  cubic 
centimeter,  in  summer  twice  as  many  or  more.  Milk  that  shows  1,000,000 
colonies  per  cubic  centimeter  or  more  is  dirty  and  has  been  carelessly  handled. 

Before  examining,  milk  must  be  thoroughly  shaken  to  evenly  distribute  the 
bacteria. 

Fermentation  tests  are  not  made  as  when  examining  water,  because  ferment- 
ing organisms  are  known  to  be  always  present,  most  of  them  are  lactic  acid  bacilli 
(non-pathogenic)  and  some  are  colon  bacilli  from  the  cow. 

Various  disease-producing  bacteria  may  be  contained  in  milk,  including 
staphylococci,  streptococci,  diphtheria  bacilli,  tubercle  bacilli  and  typhoid  bacilli; 
the  tubercle  bacillus  is  the  most  frequently  present  pathogenic  organism  and 
the  streptococcus  probably  comes  next.  From  6  to  13  per  cent,  of  all  market 
milk  contains  tubercle  bacilli. 

Many  observers  believe  that  most  of  the  epidemics  of  streptococcus  throat 
infections  are  derived  from  milk. 

It  is  practically  impossible  to  isolate  tubercle  bacilli  or  diphtheria  bacilli 
from  milk,  or  to  observe  them  in  it.  For  the  detection  of  these  organisms  in 
milk  inoculations  must  be  made,  and  these  sometimes  fail  to  disclose  them. 
Animal  inoculations  are  generally  omitted  in  the  routine  examination,  opinion 
as  to  the  bacterial  content  being  based  upon  examination  of  sediment  for  leuco- 
cytes and  bacteria,  and  plating  to  determine  the  number  of  bacteria  per  cubic 
centimeter. 

For  the  study  of  leucocytes  in  milk  and  observing  the  presence  of  staphylo- 
cocci and  streptococci,  the  best  and  quickest  is  Stewart's  method. 

When  observing  the  leucocytes  one  should  know  whether  the  milk  has  been 
heated  or  pasteurized  because  heating  apparently  increases  the  number. 

TECHNIQUE 

Shake  the  sample  to  evenly  distribute  contained  bacteria. 

Take  i  cc.  of  the  milk  and  mix  with  9  cc.  of  sterile  distilled  water,  dilution 
No.  i  equals  i  :  10;  take  i  cc.  of  dilution  No.  i  and  mix  with  99  cc.  of  water,  dilu- 
tion No.  2  equals  i  :  1000;  take  i  cc.  of  dilution  No.  2  and  mix  with  9  cc.  of  water, 
dilution  No.  3  equals  i  :  10,000. 

Take  i  cc.  of  dilution  No.  2  and  mix  with  99  cc.  of  water,  dilution  No.  4 
equals  i  :  100,000. 

176 


EXAMINATION   OF   MILK  177 

Liquefy  a  number  of  tubes  of  plain  2  to  3  per  cent,  agar  (+1.5  reaction)  and 
cool  to  45°C. 

In  each  of  the  first  two  tubes  put  i  cc.  of  i  :  1000  dilution  and  plate. 

In  each  of  the  second  two  tubes  put  0.5  cc.  of  i  :  1000  dilution  and  plate. 

In  each  of  the  third  two  tubes  put  2  cc.  of  i  :  10,000  dilution  and  plate. 

In  each  of  the  fourth  two  tubes  put  i  cc.  of  i  :  10,000  dilution  and  plate. 

In  each  of  the  fifth  two  tubes  put  0.5  cc.  of  i  :  100,000  dilution  and  plate. 

Incubate  one  set  at  room  temperature,  the  other  at  37°C.  for  3  days  and 
count  colonies  every  24  hours. 

Leucocytes. — Fill  a  Stewart's  tube  (i  cc.)  with  milk,  insert  rubber  plug  in 
one  end  and  push  the  rubber  bulb  down  tight  on  the  other.  Place  in  centrifuge 
with  plugged  end  at  periphery.  Spin  at  3000  revolutions  per  minute  for  5 
minutes.  Hold  the  tube  horizontal  and  remove  plug  without  scraping  against 
edge  of  tube.  Smear  the  sediment  collected  on  the  plug  over  an  area  of  i  square 
centimeter  on  a  glass  slide  by  rubbing  the  plug  against  it. 

Fix  by  heating  over  flame  and  stain  with  LoefBer's  methylene  blue  for  5 
minutes.  Dry  and  examine  under  oil  immersion  lens. 

The  leucocytes  are  counted  in  10  fields.  If  a  total  of  230  or  more  leucocytes 
is  observed  in  unheated  milk  it  is  condemned;  when  less  than  this  number  are 
observed  associated  with  considerable  numbers  of  streptococci  it  is  condemned. 

When  making  this  examination  many  fields  will  be  observed  in  which  there 
are  no  bacteria,  fields  that  contain  them  will  only  show  a  few — if  the  milk  is 
clean — less  than  500,000  colonies  per  cubic  centimeters  when  plated.  If  most 
of  the  fields  show  a  few  bacteria  plating  will  show  more  than  500,000  colonies 
per  cubic  centimeter,  and  if  numerous  fields  show  large  numbers  or  clumps  of 
bacteria,  the  milk  is  very  dirty  and  will  show  more  than  1,000,000  colonies  per 
cubic  centimeter. 

ANIMAL  INOCULATION  TESTS 

Place  50  cc.  of  milk  in  centrifuge  tube  and  spin  at  high  speed,  3000  revolu- 
tions per  minute  or  more,  for  10  minutes.  Remove  the  cream,  place  it  in  a 
centrifuge  tube.  Remove  the  skimmed  milk  without  disturbing  the  sediment 
in  the  bottom  of  the  tube.  Fill  both  the  centrifuge  tubes  up  to  the  5o-cc.  mark 
with  sterile  distilled  water,  shake  thoroughly  to  evenly  mix  the  contents  of  each 
tube,  then  centrifugalize  them  as  before.  Remove  the  upper  48  cc.  of  fluid 
from  each  tube  without  disturbing  the  sediment.  Suck  the  remaining  2  cc. 
of  fluid  and  sediment  from  the  cream  into  a  syringe,  and  inject  into  the  peri- 
toneal cavity  of  a  guinea-pig.  In  like  manner  inoculate  a  second  pig  with  the 
sediment  from  the  skimmed  milk. 

Healthy  pigs,  weighing  between  200  and  300  Gm.  should  be  used. 

Wholesome  milk  will  not  injure  guinea-pigs.  If  the  animals  remain  alive, 
as  they  should  if  the  milk  does  not  contain  large  numbers  of  pathogenic  organ- 
isms other  than  tubercle  bacilli,  kill  them  6  weeks  after  inoculation  and  examine 
for  tuberculosis.  It  requires  a  month  or  more  for  tubercle  bacilli  to  produce 
distinct,  characteristic  lesions  in  guinea-pigs. 


CHAPTER  III 
EXAMINATION  OF  FLUIDS  AND  SOLIDS 

Fluids  other  than  milk  are  subjected  to  bacteriological  examination  for 
several  purposes:  (i)  to  determine  whether  supposedly  sterile  fluids  are  so,  and 
if  not  the  number,  perhaps  also  the  kind,  of  bacteria  contained:  (2)  to  determine 
the  character  of  bacteria  present  in  fluids  known  to  contain  bacteria. 

TECHNIQUE 

Shake  the  sample  thoroughly,  take  a  large  drop  and  spread  it  on  a  slide  and 
fix  by  gently  heating  until  dry,  stain  with  methylene  blue  and  examine  micro- 
scopically. If  no  bacteria  are  observed  or  if  only  a  few  are  seen  in  some  fields 
and  none  in  others,  plating  will  probably  show  less  than  500,000  colonies  per 
cubic  centimeter;  if  many  bacteria  are  observed  in  most  of  the  fields,  plating 
will  show  millions  per  cubic  centimeter.  Such  a  preliminary  inspection, 
together  with  other  known  facts,  usually  give  one  an  idea  as  to  the  proper 
amount  of  dilution  required  for  plating. 

In  order  to  make  accurate  counts,  not  more  than  200  colonies  should  develop 
on  a  plate,  and  to  obtain  this,  the  richer  a  substance  is  in  bacteria  the  more  it 
must  be  diluted,  the  smaller  the  amount  placed  in  the  plate. 

To  remove  individual  colonies  from  a  plate  containing  various  organisms, 
in  order  to  further  study  the  organisms  separately  and  to  identify  them,  the 
colonies  must  be  discrete  and  sufficiently  isolated  from  each  other  to  permit  the 
removal  of  one  colony  with  a  loop,  without  touching  any  other  colony. 

Having  estimated  the  degree  of  dilution  required,  the  fluid  is  diluted  and 
plated  the  same  as  when  examining  water  or  milk. 

Plates  should  be  made  on  three  different  media,  plain  agar,  litmus  lactose 
agar  and  gelatin.  The  gelatin  plates  are  incubated  at  room  temperature,  and 
the  rest  at  37°C. 

Substances,  both  fluids  and  solids,  contaminated  with  bacteria  may  contain 
the  tetanus  bacillus.  As  this  organism  is  an  obligate  anaerobe,  several  plates 
should  be  incubated  under  anaerobic  conditions. 

If  in  addition  to  a  determination  of  the  number  of  bacteria  present,  the 
species  must  be  determined,  a  representative  plate  is  selected  after  making  the 
count,  and  one  of  each  different  kind  of  colonies  observed  is  removed  and  plated 
separately.  If  pure  cultures  develop  on  the  second  series  of  plates,  further  sub- 
cultures are  made  on  various  media  sufficient  to  establish  the  identity  of  the 
organisms.  If  the  second  series  of  plates  do  not  show  pure  cultures,  colonies 
are  removed  from  them  and  again  plated  until  a  pure  culture  is  obtained. 

Solids  are  examined  for  the  same  reasons  and  usually  in  the  same  way  as 
fluids,  except  that  they  are  first  dissolved  or  suspended  in  sterile  distilled  water. 

178 


EXAMINATION   OF   FLUIDS   AND    SOLIDS  179 

If  the  material  under  examination  is  suspected  of  being  nocuous  for  guinea- 
pigs,  2  cc.  of  it  should  be  injected  into  the  peritoneal  cavity  of  a  guinea-pig. 

EXAMINATION  OF  QUININE 

Quinine  salts  which  are  intended  for  hypodermic  administration  should  be 
examined  for  bacteria,  especially  the  tetanus  bacillus,  as  cases  of  tetanus  follow- 
ing the  hypodermic  administration  of  quinine  have  been  reported. 

TECHNIQUE 

\ 

Place  i  Gm.  of  quinine  in  each  of  two  flasks  containing  1000  cc.  of  glucose 
bouillon.  Incubate  at  37°C.  one  flask  in  an  aerobic  atmosphere,  the  other  in  an 
anaerobic  atmosphere.  Inspect  daily,  and  if  growth  occurs  centrifugalize  15  cc. 
of  the  culture  at  high  speed  for  20  minutes,  collect  the  bottom  cubic  centimeter, 
and  inject  it  subcutaneously  into  a  guinea-pig. 

Subcutaneous  injections  of  sublethal  doses  of  an  aqueous  solution  of  the 
quinine  should  also  be  made. 

EXAMINATION  OF  CAT-GUT 

Catgut  is  especially  apt  to  contain  tetanus  spores  before  treatment  and 
occasionally  does  after  attempts  at  sterilization  by  approved  methods,  hence 
cat-gut  must  always  be  proved  sterile  before  it  is  dispensed  for  use  in  surgery. 

TECHNIQUE 

Select  several  strands  or  packets  from  a  lot  and  put  in  a  large  test-tube  con- 
taining about  50  cc.  of  glucose  bouillon,  incubate  at  37°C.  in  an  anaerobic  at- 
mosphere. Inspect  daily,  and  if  growth  does  not  appear  in  5  days  it  is  sterile. 
Some  tubes  should  also  be  incubated  in  an  aerobic  'atmosphere,  and  if  desired 
guinea-pig  inoculations  may  be  made. 

EXAMINATION  OF  KETCHUP 
ESTIMATION  OF  MOLDS 

A  drop  of  the  product  to  be  examined  is  placed  on  a  microscope  slide  and  a 
cover  glass  is  placed  over  it  and  pressed  down  till  a  film  of  the  product  about  o.i 
millimeter  thick  is  obtained.  After  some  experience  this  can  be  done  fairly  well. 
A  film  much  thicker  than  this  is  too  dense  to  be  examined  successfully,  while  a 
much  thinner  film,  necessitates  pressing  the  liquids  out,  which  gives  a  very  un- 
even appearing  preparation.  When  a  satisfactory  mount  has  been  obtained, 
it  is  placed  under  the  microscope  and  examined.  The  power  used  is  about  90 
diameters,  and  such  that  the  area  of  substance  actually  examined  in  each  field  of 
view  is  approximately  1.5  square  millimeters. 

A  field  is  examined  for  the  presence  or  absence  of  mold  filaments,  the  result 
noted,  and  the  slide  moved  so  as  to  bring  an  entirely  new  field  into  view.  This 
is  repeated  till  approximately  50  fields  have  been  examined,  and  the  percentage 
of  fields  showing  molds  present  are  then  calculated.  Our  experience  has  dem- 


l8o  MEDICAL  BACTERIOLOGY 

onstrated  that  for  homemade  ketchups  this  is  practically  zero,  and  with  some 
manufactured  ketchup  it  is  as  low  as  from  2  to  5  per  cent.,  while  for  carelessly 
made  products  it  may  be  100  per  cent.;  that  is,  every  field  would  show  the 
presence  of  mold.  Investigations  under  factory  conditions  clearly  indicate  that 
with  only  reasonable  care  the  proportion  of  fields  having  molds  can  be  kept  be- 
low 25  per  cent.  A  specimen  in  which  60  per  cent,  of  the  fields  have  molds  is 
in  more  than  twice  as  bad  a  condition  as  one  containing  30  per  cent. 

After  the  percentage  reaches  30  to  40  per  cent,  it  will  be  found  that  some  of 
the  fields  frequently  have  more  than  one  filament  or  clump  of  mold,  and  the 
number  of  such  fragments  might  be  counted,  but  in  this  laboratory  this  usually 
is  not  done.  A  Thoma-Zeiss  counting  cell  with  a  center  disk  of  0.75  inch  in- 
stead of  0.25  inch,  as  usually  furnished,  would  give  a  regular  depth  of  liquid  and 
would  be  more  exact  than  the  method  described,  but  this  must  be  specially 
manufactured,  not  being  listed  in  any  of  the  catalogues  of  microscopic  supplies, 
and  the  method  as  given  is  sufficiently  accurate  for  the  purpose.  When  the 
number  of  fragments  of  mold  per  cubic  centimeter  is  estimated,  it  has  been  found 
to  range  from  virtually  zero  to  over  20,000.  There  is  no  excuse  for  a  manu- 
facturer allowing  such  conditions  to  prevail  that  his  ketchup  shows  more  than 
2000  per  cubic  centimeter,  while  some  manufacturers  by  careful  handling  hold  it 
down  to  150. 

ESTIMATION  OF  YEASTS  AND  SPORES 

Though  the  spores  referred  to  are  those  coming  from  molds  and  correspond 
to  seeds  in  more  highly  developed  plants,  it  is  frequently  very  difficult  to  differ- 
entiate some  of  them  with  certainty  from  some  yeasts  without  making  cultures, 
which  is  obviously  impossible  in  a  product  that  has  been  sterilized  by  heat. 
For  this  reason  the  yeasts  and  spores  have  been  reported  together,  and  if  there 
seemed  to  be  a  larger  percentage  of  the  latter,  mentioned  was  made  of  that  fact. 

To  make  a  count  10  cc.  of  the  product  is  thoroughly  mixed  with  20  cc.  of 
water,  and  after  being  allowed  to  rest  for  a  moment  to  permit  the  very  coarsest 
particles  to  settle  out,  a  small  drop  is  place  on  the  central  disk  of  the  Thoma- 
Zeiss  counting  cell  and  then  covered  with  a  glass.  Care  must  be  exercised  to 
have  the  slide  perfectly  clean,  so  that,  when  the  cover  glass  is  put  in  place,  a 
series  of  Newton's  rings*  results  from  the  perfect  contact  of  the  glass  surfaces; 
and,  furthermore,  the  drop  should  be  of  such  size  as  not  to  overrun  the  moat 
around  the  central  disk  and  creep  in  underneath  the  cover  glass,  thus  interfering 
with  the  contact. 

With  the  magnification  of  180,  it  has  been  the  practice  in  this  laboratory  to 
count  the  number  of  yeasts  and  spores  on  one-half  of  the  ruled  squares  on  the 
disk.  With  the  dilution  used  this  calculates  back  to  a  volume  equal  to  %o 
cubic  millimeter  in  the  original  sample,  and  reports  are  made  on  that  basis  rather 
than  on  the  number  in  a  cubic  centimeter,  because  the  former  number  is  mo- 

*  These  are  rainbow-colored  rings  produced  at  the  point  of  contact  when  polished  plates  of 
glass  are  pressed  against  each  other. 


EXAMINATION   OF   FLUIDS   AND    SOLIDS  l8l 

readily  grasped  by  the  mind  and  affords  a  simpler  notation.  To  obtain  the 
numbers  per  cubic  centimeter  the  count  made  is  simply  multiplied  by  60,000. 
It  has  been  found  in  practice  that  the  number  of  yeasts  and  spores  varies, 
for  Mo  cubic  millimeter,  from  practically  none  in  home-made  and  first-class 
commercial  ketchups  up  to  100  or  200,  and  in  one  sample  the  number  was  as  high 
as  1200.  Laboratory  experiments  show  that,  when  the  number  of  yeasts  in  raw 
pulp  reaches  from  30  to  35  in  J^o  cubic  millimeter  the  spoilage  may  frequently 
be  detectable  by  an  expert  by  odor  or  taste,  and  from  experiments  made  under 
proper  factory  conditions,  it  seems  perfectly  feasible  to  keep  the  number  in 
commercial  ketchups  below  25. 

ESTIMATION  OF  BACTERIA 

The  bacteria  are  estimated  from  the  same  mounted  sample  as  that  used  for 
the  yeasts  and  spores.  A  power  of  about  500,  obtained  by  using  a  high-power 
ocular,  is  employed  in  this  case,  and  because  of  the  greater  number  present  a 
smaller  area  is  counted  over.  Usually  the  number  in  several  areas,  each  con- 
sisting of  five  of  the  small-sized  squares,  is  counted  and  the  number  of  organisms 
per  cubic  centimeter  is  calculated  by  multiplying  the  average  number  in  these 
areas  by  2 ,400,000.  Thus  far  it  has  proved  impracticable  to  count  the  micrococci 
present,  as  they  are  likely  to  be  confused  with  other  bodies  frequently  present  in 
such  products,  such  as  particles  of  clay,  etc.  A  comparison  of  this  method  with 
the  ordinary  cultural  methods  on  samples  in  which  the  organisms  had  not  been 
killed  has  almost  invariably  shown  that  the  one  used  gives  too  low  instead  of  too 
high  results.  In  some  cases  it  was  found  to  give  not  more  than  one- third  of  the 
entire  number  present.  The  estimates  of  the  laboratory  on  this  point  may, 
therefore,  be  considered  very  conservative. 

As  regards  the  limits  which  may  be  expected  in  the  examination  of  ketchups 
for  bacteria,  it  might  be  stated  that  some  manufactured  samples  as  well  as  good, 
clean  products  made  by  household  methods,  have  been  examined,  and  the  count 
found  to  be  so  low  when  estimated  by  this  method  that  the  numbers  present  were 
reported  as  negligible.  In  other  words,  it  was  found  that  for  the  areas  counted 
over,  the  number  of  bacteria  averaged  less  than  one — that  is,  less  than 
2,400,000  per  cubic  centimeter.  It  is  unusual,  however,  for  the  final  number 
per  cubic  centimeter  to  be  less  than  from  2,000,000  to  10,000,000  organisms. 
Contrasted  with  this  number  as  a  minimum,  it  has  been  found  that  the  number 
has  occasionally  exceeded  300,000,000  per  cubic  centimeter.  Such  a  number  as 
this  would  indicate  extremely  bad  conditions  and  carelessness  in  handling,  as  the 
studies  of  factory  conditions  has  shown  that  there  is  little  excuse  for  the  number 
ever  exceeding  25,000,000  per  cubic  centimeter.  While  experiments  have  also 
shown  that  although  the  effect  produced  by  the  bacteria  on  the  product  varies 
with  different  species,  it  is  true  that  their  presence  can  frequently  be  detected 
in  the  raw  pulp  by  odor  or  taste  when  the  number  exceeds  25,000,000  per  cubic 
centimeter  and  sometimes  when  the  count  is  as  low  as  10,000,000. 

To  one  who  has  not  been  initiated  into  the  mysteries  of  the  microscope  the 
presence  of  such  a  number  of  bacteria  in  a  food  product  seems  inexcusable.  It 


1 82  MEDICAL  BACTERIOLOGY 

must  be  remembered  in  this  connection  that  most  of  these  are  probably  non- 
pathogenic  forms,  and  that  many  occur  naturally  on  the  skins  of  the  fruits. 
It  does  not  seem  just  to  set  a  standard  so  high  as  to  virtually  prohibit  the  manu- 
facture of  the  product  under  commercial  conditions;  rather,  the  idea  is  to  set  a 
limit  that  the  manufacturer  can  attain  if  due  care  is  exercised,  and  which  will 
insure  a  cleanly  product.  It  is,  however,  perfectly  possible  to  make  a  cleanly, 
wholesome  product  commercially,  even  though  the  number  of  bacteria  exceed 
that  in  the  home-made  article. 

The  allowable  limits  for  the  bacterial  content  of  tomato  pulp  vary  according 
to  the  concentration.  The  number,  however,  should  be  low  enough  so  that  when 
the  amount  of  concentrating  necessary  for  its  conversion  into  ketchup  has  been 
accomplished  the  final  product  will  still  be  within  permissible  limits  (25,000,000 
per  cubic  centimeter) .  Thus  for  a  pulp  which  must  be  concentrated  one-half  the 
bacterial  counts  should  not  exceed  about  half  the  limits  stated  above  for  the 
ketchup  itself — i.e.,  it  should  not  be  more  than  12,500,000  per  cubic  centimeter. 
The  same  general  rule  should  also  apply  to  the  content  of  molds  and  of  yeasts. 

To  insure  a  sound  product,  free  from  decay  or  any  filthy  material,  many 
factors  must  be  carefully  watched,  for  not  infrequently  oversight  in  one  par- 
ticular has  been  found  to  have  undone  the  good  effects  of  the  care  exercised  in  all 
other  ways.  Thus  it  is  possible  for  the  washing  of  the  fruit  to  be  ideal  and  the 
sorting  out  or  removing  of  the  decayed  portions  beyond  criticism,  and  yet  a 
delay  in  making  up  the  pulp  into  the  final  product  may  allow  an  amount  of 
decomposition  to  occur  which  offsets  the  care  previously  exercised.  It  has  been 
a  matter  of  surprise  to  some  manufacturers  to  find  with  what  rapidity  some  of 
these  organisms  increase.  In  one  factory  where  this  point  was  tested,  the 
bacterial  content  in  a  batch  of  tomato  trimming  juice  was  found  to  be  about 
7,000,000  per  cubic  centimeter  when  taken  from  the  peeling  tables,  and  after 
standing  at  room  temperature  for  5  hours  it  had  increased  to  84,000,000.  This 
was  a  twelvefold  increase  in  a  length  of  time  which  was  less  than  half  the  working 
day  for  some  of  the  factories  visited.  At  the  end  of  5  days  the  number  had  in- 
creased to  nearly  3,000,000,000  per  cubic  centimeter.  Thus  it  is  seen  that  delay 
in  manufacture  is  very  liable  to  result  disastrously. 

Such  facts  as  these  serve  to  emphasize  the  great  importance  of  absolute 
cleanliness  in  every  detail  about  factories  of  this  kind.  Dirty  floors  and  ceilings 
and  apparatus  left  with  residues  of  tomato  product  clinging  to  them  are  most 
fruitful  sources  for  the  contamination  of  new  batches  of  the  product.  To  clean 
such  an  establishment  properly  it  is  almost  imperative  that  machinery  and  wood- 
work be  washed  by  means  of  live  steam  used  lavishly  at  frequent  intervals.  To 
leave  buckets,  tables,  conveyors,  or  any  other  part  of  the  equipment  or  floors 
overnight  without  cleansing  them,  as  was  the  practice  in  some  factories,  is 
reprehensible  and  tends  to  contaminate  the  product  and  lead  to  spoilage  and 
loss.* 

*  From  Bulletin,  Feb.  12,  1911,  U.  S.  Department  of  Agriculture,  "Tomato  Ketchup  Under 
the  Microscope,"  by  B.  J.  Howard,  Chief,  Microchemical  Laboratory. 


EXAMINATION    OF   FLUIDS   AND    SOLIDS  183 

EXAMINATION  OF  EGGS 

When  laid,  eggs  contain  a  few  bacteria;  if  the  shells  have  no  cracks  or  breaks 
in  them  and  are  free  of  macroscopic  particles  of  dirt  and  if  such  eggs  are  collected 
within  several  hours  after  they  have  been  laid  and  immediately  stored  in  clean, 
dry  containers  at  about  i5°C.,  the  eggs  remain  "fresh"  for  many  weeks  with 
slight,  if  any,  multiplication  of  contained  bacteria. 

Bacteria  rapidly  increase  in  cracked  eggs  and  those  exposed  to  a  temperature 
of  3o°C.  to  4o°C. 

Market  eggs,  shown  by  candling,  chemical  examination  and  diet  tests,  to  be 
palatable  and  nutritious,  yield  20,000  to  200,000  colonies  per  gram  when  cultured 
a  small  portion  of  which  are  colon  bacilli. 

Eggs  in  the  shell  that  show  more  than  1000  colonies  per  gram  are  not  accept- 
able as  food. 

Frozen  eggs  and  desiccated  eggs  (either  whites,  yolks  or  whole  eggs)  always 
show  a  relatively  more  abundant  flora  than  eggs  in  the  shell. 

Frozen  and  desiccated  eggs  may  be  considered  acceptable  when  the  cultural 
test  shows  less  than  2,000,000  colonies  per  gram,  npt  more  than  0.02  per  cent,  of 
which  are  colon  bacilli.  When  the  percentage  of  colon  bacilli  is  considerably 
greater  or  when  the  total  count  is  above  20,000,000  colonies  per  cubic  centi- 
meter, the  eggs  have  been  handled,  canned  or  stored  in  a  manner  that  makes 
them  dangerous  foodstuff  and  when  the  count  is  above  20,000,000  hi  addition 
to  being  dangerous  to  health,  they  have  been  robbed  of  a  considerable  portion 
of  their  nutritive  value. 

These  figures  and  conclusions  .are  based  on  a  resume  of  the  extensive  original 
investigations  of  R  .  C.  Rosenberger. 

TECHNIQUE  FOR  EXAMINATION  OF  EGGS  IN  SHELL 

1.  Wash  the  eggs  in  5  per  cent,  phenol  solution  at  5o°C.  by  gently  sponging 
with  cotton. 

2.  Rinse  in  sterile  water. 

3.  Break  shell  with  sterile  spatula  and  empty  contents  into  wide-mouthed, 
sterile,  weighed  Erlenmeyer  flask. 

4.  Determine  weight  of  egg  or  eggs  in  flask  and  add  an  equal  volume  of 
sterile  water,  shake  until  homogenized. 

5.  Put  i.o  Gm.  in  flask  and  mix  with  499  Gm.  of  sterile  water  (i  :  1000  di- 
lution). 

6.  Mix  i  cc.  of  the  i  :  1000  dilution  with  9  cc.  of  sterile  water  and  plates  of 
plain  agar  and  litmus  lactose  agar  with  the  following  quantities:  o.i  cc.,  o.2cc., 
0.5  cc.,  0.7  cc.  and  i.o  cc.;  also  plant  o.i  cc.,  0.5  cc.  and  i.o  cc.  in  fermentation 
tubes  containing  litmus  lactose  broth. 

Incubate  at  37°C.  for  3  days  and  inspect  each  day.  Subculture  representa- 
tive red  colonies  that  appear  on  litmus  agar  and  make  differential  tests  for  iden- 
tification of  colon  bacillus. 


184  MEDICAL  BACTERIOLOGY 

FROZEN  EGGS  AND  DESICCATED  EGGS 

1.  Weigh  out  i.o  Gm.  of  egg  on  a  sterile  watch  crystal,  put  in  sterile  flask 
with  99  cc.  of  sterile  water,  shake  until  entirely  dissolved  and  thoroughly  mixed. 

2.  Transfer  i  cc.  of  first  dilution  (i  :  100)  to  a  second  flask  containing  99  cc. 
of  sterile  water  and  thoroughly  mix,  making  a  i  :  10,000  dilution. 

3.  Plant  both  plain  agar  and  litmus  lactose  agar  plates  with  the  following 
quantities  of  the  second  dilution:  o.i  cc.,  0.2  cc.,  0.5  cc.,  0.7  cc.  and  i.o  cc. 

4.  Incubate,  inspect  and  count  as  when  examining  eggs  taken  from  shell. 


CHAPTER  IV 

DETERMINATION  OF  THE  GERMICIDAL  POWER  OF  CHEMICAL 

DISINFECTANTS 

To  accurately  determine  the  amount  of  a  particular  chemical  required  to 
kill  all  pathogenic  bacteria,  under  varying  conditions  of  environment,  is  prac- 
tically impossible.  The  germicidal  power  of  a  chemical  disinfectant  varies  in 
different  media  and  at  different  temperatures;  some  which  are  powerful  germi- 
cides in  media  free  of  albumin  are  nearly  inert  when  associated  with  albumin; 
some  are  powerful  germicides  when  in  solution  and  less  germicidal  when  mixed 
with  a  substance  that  throws  them  out  of  solution. 

An  estimation  of  the  germicidal  power  of  a  given  substance  may  be  made 
which  gives  valuable  information  regarding  the  proper  employment  of  that  sub- 
stance as  a  disinfectant  under  various  conditions. 

This  is  done  by  obtaining  typical  pure  cultures  of  as  many  different  organ- 
isms as  possible — staphylococci,  typhoid,  colon,  diphtheria,  tubercle,  anthrax 
and  tetanus  bacilli,  etc. — mixing  different  amounts  of  the  disinfectant  with  a 
fixed  amount  of  the  bacteria,  both  with  and  without  the  admixture  of  organic 
matter,  letting  the  mixture  stand  at  a  given  temperature  for  a  certain  time  and 
then  testing  the  mixture  for  sterility  by  making  subcultures  from  it. 

This  is  a  very  extensive,  time-consuming  procedure  and  at  best  yields  only 
approximate  results.  Different  strains  of  the  same  organism  under  identical 
conditions  often  show  variations  of  several  hundred  per  cent,  in  their  resistance 
to  a  chemical  disinfectant. 

Within  certain  limits  the  following  conditions  influence  the  action  of  chemical 
disinfectants: 

1.  Length  of  time  the  disinfectant  is  in  contact  with  bacteria. 

2.  Temperature  at  which  the  contact  occurs  (at  low  temperatures  disin- 
fectants are  much  less  active  than  at  high,  for  every  rise  of  10°  the  efficiency  is 
increased  two-  to  tenfold. 

3.  Physical  and  chemical  properties  of  the  medium  in  which  the  infective 
organisms  exist. 

4.  The  amount  of  disinfectant  and  the  number  and  nature  of  the  bacteria. 
On  account  of  the  many  factors  that  complicate  and  limit  approximate 

determinations  of  the  disinfectant  power  of  chemicals,  such  determinations  were 
largely  neglected  until  recent  years  with  a  consequent  development  of  gross 
misconceptions  and  inefficient  use  of  chemical  disinfectants. 

In  1898  Rideal  and  Walker  introduced  a  test,  known  as  the  "  Rideal- Walker 
Carbolic  Acid  Coefficient  Test,"  for  the  determination  of  the  relative  disinfect- 
ant power  of  chemicals,  in  comparison  with  that  of  carbolic  acid.  Later  this 
test  was  improved  upon  by  the  introduction  of  one  known  as  the  "Lancet 

185 


1 86  MEDICAL  BACTERIOLOGY 

Method,"  and  still  later  Anderson  and  McClintic  introduced  the  "Hygienic 
Laboratory  Method." 

None  of  these  methods  is  ideal;  they  are  difficult  and  time-consuming  and 
yield  only  approximate  results,  yet  they  are  of  great  value  as  approximate  and 
relative  knowledge  of  disinfectant  value  is  by  far  superior  to  total  ignorance. 

Of  the  various  methods,  the  "Hygienic  Laboratory  Method"  is  unquestion- 
ably the  best;  especially  in  the  United  States  it  should  always  be  the  method 
of  choice.  In  "Hygienic  Laboratory  Bulletin,  No.  82,  of  the  Public  Health 
and  Marine  Hospital  Service  of  the  United  States,"  April,  1912,  Anderson  and 
McClintic  describe  their  test  in  a  clear,  concise  manner.  Those  intending  to 
make  coefficient  tests  should  obtain  the  above-mentioned  bulletin.  The  follow- 
ing description  of  the  tests  is  quoted  from  Anderson  and  McClintic: 

PRINCIPAL  FACTORS  INVOLVED   IN  THE  EXAMINATION   OF  DISINFECTANTS 

"Lack  of  attention  to  the  different  factors  concerned  in  the  examination  of 
disinfectants  is  responsible  for  a  large  percentage  of  the  inconsistencies  or  dis- 
crepancies in  results  obtained  by  the  same  or  by  different  workers  when  working 
with  the  same  disinfectants.  Unless  strict  attention  is  paid  to  the  various  in- 
fluences involved  it  is  useless  to  expect  to  find  any  method  satisfactory. 

In  order  to  better  emphasize  the  effect  of  these  influences  upon  the  results 
obtained,  the  various  factors  involved  will  be  discussed  under  the  appropriate 
headings. 

TEST  ORGANISM 

Unless  different  observers  use  the  same  species  of  organism  there  can  be  no 
possibility  of  uniformity  in  results.  The  coefficient  obtained  with  different 
species  may  vary  as  much  as  300  per  cent.  For  this  reason  it  is  important  that 
one  species  be  selected  for  use  as  the  test  organism.  It  would  be  highly  desir- 
able if  the  same  strain  of  this  species  could  be  used  by  all  workers  in  the 
testing  of  disinfectants,  as  there  is  often  a  variation  in  the  resistance  of  dif- 
ferent strains  of  the  same  species.  This  objection  does  not  apply  as  much  to 
the  typhoid  organism  as  to  the  colon  bacillus,  and  to  some  other  bacteria. 

We  made  a  number  of  comparative  tests  with  different  strains  of  B.  typhosus 
and  B.  coli  and  found  a  very  much  greater  difference  in  the  resistance  of  differ- 
ent strains  of  the  colon  bacillus  than  of  the  typhoid  bacillus. 

It  is  most  important  that,  before  being  used  for  a  test,  the  organism  be  car- 
ried over  on  broth  daily  for  at  least  i  week.  In  all  cases  a  24-hour  culture 
should  be  used,  as  there  is  decided  difference  in  the  resistance  of  a  24-hour  and 
a  48-hour  culture,  the  latter  being  the  more  resistant. 

In  order  to  avoid  clumps  in  the  culture  the  24-hour  broth  culture  should  be 
well  shaken  and  then  filtered  through  sterile  filter-paper  into  a  sterile  test-tube. 
After  this  it  should  be  placed  in  the  water  bath  in  order  that  it  may  reach  the 
standard  temperature  before  being  added  to  the  disinfectant  dilutions. 


GERMICIDAL   POWDER    OF    CHEMICAL   DISINFECTANTS  187 

TEMPERATURE  OF  EXPERIMENT 

It  is  a  well-known  principle  in  the  use  of  disinfectants  that,  within  certain 
limits,  the  higher  the  temperature  at  which  the  disinfectant  is  used  the  greater 
are  its  germicidal  properties.  This  increase  in  the  germicidal  properties  of  dis- 
infectants through  the  influence  of  heat  is  not  the  same  for  all  disinfectants; 
some,  such  as  formaldehyde,  are  more  strongly  influenced  than  others.  It  will 
be  seen  that  at  i5°C.  phenol,  in  dilution  of  i  :8o,  killed  the  typhoid  organism 
in  2^j  minutes,  while  at  a  temperature  of  3o°C.  the  organism  was  killed  in  the 
same  length  of  time  by  a  dilution  of  i  :  1 20. 

On  account  of  the  great  variation  of  temperature  in  the  United  States,  espe- 
cially during  the  summer,  it  becomes  necessary  that  a  standard  temperature 
be  adopted.  We  have  adopted  a  temperature  of  2o°C.  and  have  devised  a  sim- 
ple water  bath  to  be  used  for  maintaining  this  temperature.  This  bath  consists 
of  a  wooden  box  20  inches  deep,  21  inches  long,  and  21  inches  wide.  Inside  this 
box  a  i4-quart  agateware  pail  10  inches  deep  is  placed  and  sawdust  is  well- 
packed  around,  sufficient  being  placed  on  the  bottom  of  the  box  to  bring  the 
rim  of  the  pail  on  a  level  with  the  top  of  the  box. 

A  tightly  fitting  wooden  cover  is  placed  over  the  pail,  so  made  that  the  edges 
project  slightly  over  the  rim.  In  the  cover  are  a  sufficient  number  of  holes  for 
the  seeding  tubes,  a  thermometer,  and  the  tube  containing  the  culture.  About 
3  inches  below  the  rim  of  the  pail  a  false  bottom  of  wire  gauze  is  placed;  this  is 
for  the  seeding  tubes,  etc.,  to  rest  on.  Water  is  placed  in  the  pail  to  within 
%  inch  of  the  top. 

When  an  experiment  is  to  be  made  the  temperature  of  the  water  in  the  pail 
is  taken,  and  if  above  or  below  2o°C.  it  is  brought  to  the  desired  temperature 
by  the  addition  of  either  cold  or  hot  water.  It  will  be  found  that  only  very 
slight  change  takes  place  in  the  temperature  of  the  bath  in  an  hour  and  that  it 
is  an  easy  matter  to  keep  the  temperature  of  the  bath  at  the  figures  desired. 
It  is  of  advantage,  in  regulating  the  temperature  of  the  bath,  to  have  a  spigot 
in  the  bottom  of  the  pail  to  draw  off  the  water  when  so  desired. 


PROPORTION  OF  CULTURE  TO  DISINFECTANT 

As  disinfection  is  the  result  of  chemical  action  of  the  disinfecting  agent  upon 
the  test  organism,  mass  action  is  an  important  factor  in  the  testing  of  disinfect- 
ants. By  this  is  meant  that  within  certain  limits  the  greater  the  number  of 
bacteria  added  to  the  disinfectant  dilution  the  stronger  the  dilution  required 
to  do  the  same  work.  For  this  reason  it  is  important  that  the  amount  of  culture 
to  be  added  to  the  dilution  should  be  stated  in  definite  quantities  and  not  in 
"drops"  or  in  " spoonfuls."  We  have  adopted  the  practice  of  using  o.i  cc.  of 
a  24-hour  broth  culture.  For  measuring  this  we  use  a  delivery  pipette  gradu- 
ated in  tenths. 


1 88  MEDICAL  BACTERIOLOGY 


MEDIA  FOR  SUBCULTURES 

There  is  probably  no  one  factor,  with  the  possible  exception  of  temperature, 
that  has  more  to  do  with  irregularities  in  results  than  the  media  for  subcultures. 
Where  the  typhoid  bacillus  is  used  for  the  test  organism,  as  in  the  Rideal- Walker 
method,  and  the  method  proposed  by  us,  it  is  of  paramount  importance  that  the 
media  have  a  reaction  of  just +1.5.  A  reaction  greater  than  this  exerts  a 
decided  inhibiting  action  upon  the  growth  of  the  transplanted  organism.  This 
is  an  important  point,  for  if  the  transplant  is  made  from  a  test  dilution  which  is 
just  under  the  killing  strength  of  the  disinfectant,  the  inhibiting  action  of  the 
media  may  be  sufficient  to  prevent  growth,  thus  giving  a  false  result.  In  the 
hands  of  different  workers  a  difference  in  the  reaction  of  the  media  may  result 
from  the  degree  to  which  the  color  reaction  in  titration  is  carried.  We  always 
carry  it  to  the  point  where  the  pink  color  is  distinctly  perceptible,  but  even 
then  there  seems  to  be  at  times  a  slight  difference  in  various  lots  of  our  media. 

It  is  a  noteworthy  fact  that  the  influence  of  the  reaction  of  the  subculture 
media  upon  the  growth  of  the  exposed  organism  was  decidedly  more  pronounced 
after  it  had  been  exposed  to  phenol  than  to  any  of  the  other  disinfectants  tried. 

It  was  found,  too,  that  a  more  vigorous  growth  and  a  growth  from  stronger 
solutions  were  obtained  when  the  exposed  organisms  were  planted  in  meat 
broth  than  when  they  were  planted  in  extract  broth.  It  is  therefore  evident 
from  the  above  that  the  reaction  and  character  of  the  subculture  media  has  an 
important  bearing  upon  the  results  obtained  in  determining,  the  phenol  coeffi- 
cient of  disinfectants.  However,  as  extract  broth  is  more  uniform  in  composi- 
tion, more  easily  prepared,  and  cheaper  than  meat  broth,  we  recommend  that 
extract  broth  be  always  used  and  when  it  is  not,  that  the  fact  be  so  stated. 

The  amount  of  media  in  the  tubes  for  subculture  should  be  sufficient  to 
prevent  any  antiseptic  action,  due  to  the  transferred  disinfectant.  With  some 
substances,  such  as  bichloride  of  mercury,  this  is  often  an  important  point. 

It  may  be  stated  here  that  in  our  work  with  some  disinfectants,  particularly 
those  containing  coal-tar  products,  the  disinfectant  carried  over  in  making  the 
inoculations  of  the  subcultures  caused  a  distinct  cloudiness  in  the  media;  but 
after  48  hours'  incubation  this  always  cleared  up  so  that  there  was  no  difficulty 
in  making  out  the  presence  or  absence  of  growth. 

MacConkey's  bile-salt  medium  was  given  a  limited  trial  with  B.  coli  ct>m- 
munis.  We  found  that  after  exposing  the  B.  coli  communis  to  the  action  of  a 
i  per  cent,  solution  of  carbolic  acid  and  planting  in  MacConkey's  medium  and 
extract  broth,  respectively,  every  2^  minutes  for  15  minutes,  and  incubating 
for  48  hours,  there  was  a  growth  in  all  the  tubes  of  the  extract  broth,  but  only  in 
a  2j^-minute  tube  of  MacConkey's  medium.  This  condition  or  result  was 
more  marked  with  carbolic  acid  than  with  any  other  disinfectant  tried. 

When  using  the  B.  typhosus  the  possibility  of  contamination  in  the  tubes  of 
broth  that  show  a  growth  at  the  end  of  48  hours  can  be  determined  by  the  use  of 
antityphoid  serum. 


GERMICIDAL   POWER   OF   CHEMICAL   DISINFECTANTS  189 


ORGANIC  MATTER 

Under  practical  conditions  disinfectants  are  commonly  used  in  the  presence 
of  more  or  less  organic  matter.  It  is  therefore  very  important  to  know  to  what 
extent  the  germicidal  efficiency  of  a  disinfectant  is  affected  by  the  presence  of 
organic  matters. 

The  character  and  quality  of  organic  matter  present  as  well  as  its  effect  upon 
the  germicidal  efficiency  of  different  disinfectants  are  such  widely  varying 
factors  that  the  standardization  of  disinfectants  in  the  presence  of  organic 
matter  is  a  rather  difficult  problem.  We  have  done  a  great  deal  of  experimental 
work  with  disinfectants  in  which  various  kinds  of  organic  matter  were  tried, 
and  it  has  been  difficult  to  find  a  form  of  organic  matter  that  is  entirely  satis- 
factory for  standardization  purposes.  Urine,  blood  serum,  dead  (killed  by 
heat)  broth  cultures  of  the  typhoid  bacillus,  peptone,  gelatin,  egg  albumen, 
starch,  etc.,  were  all  given  a  trial  and  we  have  finally  decided  to  use  a  mixture 
of  an  aqueous  solution  of  peptone  and  gelatin. 

It  is  manifestly  necessary  that  any  form  of  organic  matter,  used  for  stand- 
ardization of  disinfectants,  be  of  known,  definite  composition.  Unless  this  is 
the  case  only  widely  varying  results  can  be  expected.  A  mixture  of  urine  and 
feces  would  probably  simulate  the  natural  conditions  under  which  disinfectants 
are  ordinarily  used  more  closely  than  any  of  the  other  forms  of  organic  matter, 
but  the  organic  content  of  different  specimens  of  urine  and  feces  is  so  variable 
as  to  practically  exclude  their  use  in  standardization  work.  Blood  serum  is 
constant  in  composition,  but  it  is  often  difficult  to  obtain,  particularly  in  a 
sterile  condition,  and,  of  course,  organic  matter  must  be  sterile  when  it  is  used. 

The  results  obtained  with  starch  and  with  egg  albumen  used  as  organic 
matter  were,  on  the  whole,  unsatisfactory. 

Dead  broth  cultures  of  the  typhoid  bacillus  and  the  peptone-gelatin  mixture 
gave  rather  similar  results,  but  on  account  of  the  ease  with  which  the  latter  can 
be  procured  and  prepared  we  have  chosen  it  for  use  in  our  work.  Peptone  and 
gelatin  are  fairly  constant  in  composition,  and  a  mixture  of  the  two  can  be 
sterilized  without  altering  the  composition  of  either  the  peptone  or  the  gelatin. 

HYGIENIC  LABORATORY  PHENOL  COEFFICIENT 
A.  Without  Organic  Matter 

Having  discussed  the  necessity  for  a  satisfactory  method  of  standardizing 
disinfectants  and  the  factors  involved  in  the  examination  of  disinfectants,  we 
present  below  the  method  we  have  devised. 

When  this  method  is  used  for  the  standardization  of  disinfectants  we  recom- 
mend that  it  be  referred  to  as  the  "Hygienic  Laboratory  phenol  coefficient." 

We  prefer  to  use  the  word  "phenol"  instead  of  "carbolic  acid"  when  speak- 
ing of  the  coefficient,  especially  since  certain  dealers  advertise  for  sale  carbolic 
acids  which  vary  greatly  in  the  proportion  of  phenol  present. 


I QO  MEDICAL  BACTERIOLOGY 


MEDIA 

Standard  extract  broth  is  used,  both  for  the  culture  to  be  tested  and  for  the 
subcultures  made  after  exposure  to  the  disinfectant.  The  broth  is  made  from 
Liebig's  extract  of  beef  and  is  in  exact  accordance  with  the  standard  methods 
adopted  by  the  American  Public  Health  Association  for  water  analysis.  Ten 
cubic  centimeters  of  the  broth  are  put  into  each  test-tube.  This  amount  of 
broth  has  been  found  sufficient  to  avoid  any  antiseptic  action  of  the  disin- 
fectant carried  over.  It  is  important  that  the  reaction  of  the  media  is  just 


ORGANISM 

For  the  test  organism  a  24-hour-old  broth  culture  in  extract  broth  of  the 
B.  typhosus  (Hopkins)  is  used.  Before  beginning  a  test  the  culture  should  be 
carried  over  every  24  hours  on  at  least  3  successive  days.  For  carrying  over 
the  culture  one  loopful  of  a  4-millimeters  platinum  loop  is  used. 

Before  being  added  to  the  disinfectant  the  culture  is  well  shaken,  filtered 
through  a  sterile  filter-paper,  and  placed  in  the  water  bath  in  order  that  it  may 
reach  a  temperature  of  2o°C.  before  being  added  to  the  disinfectant. 


TEMPERATURE 

A  standard  temperature  of  2O°C.  has  been  adopted  for  all  experiments. 
This  temperature  is  obtained  by  the  use  of  a  specially  devised  water  bath.  The 
culture  and  dilutions  of  the  disinfectant  are  brought  to  this  temperature  before 
the  beginning  of  the  test. 

PROPORTION  OF  CULTURE  TO  DISINFECTANT 

One-tenth  cubic  centimeter  of  the  culture  is  used,  added  to  5  cc.  of  the  dis- 
infectant dilution.  The  amount  of  culture  is  measured  with  a  pipette  grad- 
uated in  tenths  of  a  cubic  centimeter. 

INOCULATION  LOOPS 

For  making  the  transfer  of  the  culture  after  exposure  to  the  disinfectant  a 
platinum  loop  4  millimeters  in  diameter  of  23  United  States  standard  gauge 
wire  is  used.  We  have  found  it  is  of  advantage  to  have  at  least  four,  and  pref- 
erably six,  loops.  In  order  to  save  time  in  flaming  the  following  method  was 
devised: 

A  block  about  3  inches  wide,  10  inches  high,  and  12  inches  long,  containing 
four  or  six  grooves,  spaced  2  inches  apart,  is  used.  Into  each  of  the  grooves 


GERMICIDAL  POWER   OF   CHEMICAL  DISINFECTANTS  IQI 

the  platinum  loop  is  laid  so  that  the  end  of  the  loops  extend  about  5  inches 
beyond  the  side  of  the  block.  The  first  step  in  the  operation  is  to  sterilize  each 
loop  by  flaming  with  a  fantail  Bunsen  burner  before  beginning  the  experiment. 
When  ready  to  begin  the  operation  the  loop  farthest  from  the  operator  is 
taken  in  the  right  hand  and  the  inoculation  made.  It  is  then  replaced  in  the 
groove  with  the  right  hand  and  the  Bunsen  burner  (fantail)  placed  under  it 
with  the  left  hand.  The  next  loop  is  then  used,  replaced  in  its  groove,  and  the 
Bunsen  burner  placed  under  it  with  the  left  hand,  the  first  loop  having  been 
heated  to  redness  while  the  second  loop  was  in  use.  This  procedure  is  then 
continued  until  all  the  inoculations  have  been  made.  The  time  required  in 
making  the  inoculations  and  in  replacing  the  loop  is  short,  it  being  found  that 
15  seconds  is  ample. 

INCUBATION 

The  subcultures  are  incubated  48  hours  at  37°C.,  and  the  results  then  read 

off  and  tabulated. 

i 

DILUTIONS 

Capacity  pipettes  for  the  original  dilutions  are  invariably  used  for  the  phenol 
controls  a  standard  dilution  of  pure  phenol  ("Merck's  Silver  Label")  is  made 
and  standardized  by  the  U.  S.  P.  method  (Koppeschaar)  to  contain  exactly 
5  per  cent,  of  pure  phenol  by  weight.  From  this  stock  solution  the  higher  dilu- 
tions are  made  fresh  each  day  for  that  day's  test. 

For  the  dilutions  of  the  disinfectant  a  5  per  cent,  solution  is  made  by  adding 
5  cc.  of  the  disinfectant  to  95  cc.  of  sterile  distilled  water.  A  standardized 
5  cc.  capacity  pipette  is  used  for  this,  and  after  filling  the  pipette  is  wiped  off 
with  sterile  gauze.  The  contents  of  the  pipette  are  then  delivered  into'a  cylinder 
containing  95  cc.  of  sterile  distilled  water,  and  the  pipette  washed  out  as  clean 
as  possible  by  aspiration  and  blowing  out  the  contents  of  the  pipette  into  the 
cylinder.  The  contents  of  the  cylinder  are  then  thoroughly  shaken  and  the 
dilutions  up  to  i :  500  made  from  it,  using  delivery  pipettes  for  measuring.  For 
those  disinfectants  which  do  not  readily  form  a  5  per  cent,  solution  we  make 
a  i  per  cent,  stock  solution,  and  from  this  make  the  dilutions  greater  than 
1:100  in  accordance  with  the  second  table  of  dilutions.  If  greater  dilutions 
than  i :  500  are  to  be  made,  a  i  per  cent,  solution  is  made  from  the  5  per  cent, 
solution,  and  the  higher  dilutions  made  from  this. 

We  have  adopted  the  following  scale  for  making  dilutions: 

For  dilutions  up  to  i :  70,  increase  or  decrease  by  a  difference  of  5  (i.e.,  parts 
of  water). 

From  i :  70  to  i :  160  by  a  difference  of  10. 

From  i :  160  to  i :  200  by  a  difference  of  20. 

From  i :  200  to  1 1400  by  a  difference  of  25. 

From  i :  400  to  i :  900  by  a  difference  of  50. 


MEDICAL  BACTERIOLOGY 

From  i :  900  to  i :  1800  by  a  difference  of  100. 

From  i :  800  to  1 13200  by  a  difference  of  200. 
And  so  on  if  higher  dilutions  are  necessary. 

It  is  important  that  the  cylinders  used  for  making  the  dilutions  be  correctly 
graduated,  as  we  have  found  disregard  of  this  factor  an  important  source  of 
error.  It  is  preferable  to  use  standardized  cylinders  and  pipettes,  and  we  recom- 
mend that  they  be  used  whenever  possible.  They,  of  course,  should  be  per- 
fectly clean.  For  making  the  dilutions  in  accordance  with  the  above  scheme 
we  have  found  the  following  tables  of  much  service: 

TABLE  15  (FOR  DILUTIONS). — STOCK  5  PER  CENT.  SOLUTION 
(5  cc.  disinfectant  +  95  cc.  distilled  water  =  solution  A) 


Cc. 

of  A 

Cc.  of 

dist. 

Cc. 

of  A 

Cc.  of 
dist. 

Cc.      Cc.  of 
of  A       dist. 

water 

water 

water 

1:20 

= 

20  + 

O 

or 

10  — 

O 

or 

4+0 

1:25 

= 

2O  + 

5 

or 

10  + 

2K 

or 

4+     I 

1:30 

= 

20  + 

IO 

or 

IO  + 

5 

or 

4+     2 

1:35 

= 

2O  + 

15 

or 

IO  + 

7^ 

or 

4+    3 

1:40 

= 

2O  + 

20 

or 

10  + 

IO 

or 

4+4 

i:45 

= 

2O  + 

25 

or 

IO  + 

I2>2 

or 

4+    5 

i:50 

= 

2O  + 

30 

or 

10  + 

15 

or 

4+    6 

i:55 

= 

20  + 

35 

or 

IO  + 

I7/^ 

or 

4+    7 

1:60 

= 

2O  + 

40 

or 

IO  + 

2O 

or 

4+    8 

1:65 

= 

20  + 

45 

or 

IO  + 

22^ 

or 

4+9 

1:70 

= 

2O  + 

50 

or 

IO  + 

25 

or 

4  +  10 

1:70 

= 

2O  + 

50 

or 

10  + 

25 

or 

4+10 

1:80 

= 

20  + 

60 

or 

IO  + 

30 

or 

4  +  12 

1:90 

= 

2O  + 

70 

or 

IO  + 

35 

or 

4  +  14 

i:  100 

= 

20  + 

80 

or 

IO  + 

40 

or 

4+16 

i:no 

= 

2O  + 

90 

or 

IO  + 

45 

or 

4  +  18 

i:  1  20 

= 

2O  + 

IOO 

or 

10  + 

50 

or 

4+20 

i:  130 

= 

20  + 

no 

or 

IO  + 

55 

or 

4+22 

i:  140 

= 

2O  + 

1  20 

or 

IO  + 

60 

or 

4+24 

i:  150 

= 

20  + 

130 

or 

10  + 

65 

or 

4+26 

1:160 

= 

2O  + 

140 

or 

IO  + 

70 

or 

4+28 

i:  160 

= 

2O  + 

140 

or 

10  + 

70 

or 

4+28 

1:180 

= 

20  + 

160 

or 

IO  + 

80 

or 

4  +  32 

1:200 

= 

2O  + 

1  80 

or 

IO  + 

90 

or 

4  +  36 

i:  200 

= 

2O  + 

1  80 

or 

4  + 

36 

or 

2   +   18 

1:225 

= 

2O  + 

1  80 

or 

4  + 

41 

or 

2  +  20^ 

1:250 

= 

2O  + 

230 

or 

4  + 

46 

or 

2   -   23 

1:275 

= 

20  + 

255 

or 

4  + 

51 

or 

2  +  25K 

1:300 

= 

2O  + 

280 

or 

4  + 

56 

or 

2  +  28 

1:325 

= 

2O  + 

305 

or 

4  + 

61 

or 

2  +  30^ 

1:350 

= 

20  + 

330 

or 

4  + 

66 

or 

2  +  33 

i:375 

= 

2O  + 

355 

or 

4  + 

7i 

or 

2  +  35^ 

1:400 

= 

2O  + 

380 

or 

4  + 

76 

or 

2  +38 

1:450 

= 

2O  + 

430 

or 

4  + 

86 

or 

2  +  43 

1:500 

= 

2O  + 

480 

or 

4  + 

96 

or 

2  +48 

GERMICIDAL   POWER   OF   CHEMICAL   DISINFECTANTS 

TABLE  16  (FOR  DILUTIONS). — STOCK  i  PER  CENT  SOLUTION 
[i  cc.  disinfectant  +  99  cc.  distilled  water  =  solution  B] 


193 


i: 
i: 
i  : 
i  : 
i: 
i: 
i: 
i: 
i: 

IOO 

no 
1  20 
130 
140 
150 
1  60 
1  60 
1  80 

= 

Cc. 

of  A 

IOO  -} 
IOO  -| 
IOO  H 
IOO  -\ 

loo  H 

IOO  H 
IOO  H 
IOO  H 
IOO  H 

Cc.  of 
dist. 
water 

0 

-  10 

-   20 
-   30 
-   40 
-   50 
-   60 
-   60 
-   80 

or 
or 
or 
or 
or 
or 
or 
or 
or 

Cc. 

of  A 

10  + 

IO  + 
10  + 
10  + 
10  + 
10  + 
10  + 
10  + 
10  + 

Cc.  of 

dist. 
water 

o 

I 

2 

3 
4 
5 
6 
6 
8 

Cc    Cc-  o£ 
0VC:    dist. 

of  A    water 

i: 

200 

= 

ioo  H 

-  ioo 

or 

IO 

+ 

IO 

i  : 

200 

= 

IOO  H 

-  IOO 

or 

IO 

+ 

IO 

or 

4  H 

-  4 

i: 

225 

= 

IOO  H 

-125 

or 

10 

+ 

12^2 

or 

4  H 

-  5 

i: 

250 

= 

IOO  H 

-  150 

or 

IO 

+ 

IS 

or 

4  H 

-  6 

i  : 

275 

= 

IOO  H 

-  175 

or 

IO 

+ 

I?K 

or 

4  H 

r  7 

i: 

300 

= 

IOO  H 

-  200 

or 

10 

+ 

20 

or 

4  H 

-  8 

i: 

325 

= 

IOO  H 

-  225 

or 

IO 

+ 

22^ 

or 

4  H 

-  9 

i: 

350 

= 

IOO  H 

-  250 

or 

10 

+ 

25 

or 

4  H 

-  10 

i: 

375 

= 

IOO  H 

-  275 

or 

IO 

+ 

27^2 

or 

4H 

-  ii 

i: 

400 

= 

IOO  H 

-  300 

or 

IO 

+ 

30 

or 

4  H 

-  12 

i: 

400 

= 

10  H 

-   30 

or 

4 

+ 

12 

or 

2  -\ 

-  6 

i: 

450 

= 

10  H 

r  35 

or 

4 

+ 

14 

or 

2  H 

-  7 

i: 

500 

= 

10  H 

h  40 

or 

4 

+ 

16 

or 

.   2  H 

-  8 

i: 

500 

= 

10  - 

h  45 

or 

4 

+ 

18 

or 

2  H 

-  9 

i: 

600 

= 

IO  - 

H  50 

or 

4 

+ 

20 

or 

2  H 

-  10 

i  : 

650 

= 

10  - 

h  55 

or 

4 

+ 

22 

or 

2  H 

r  ii 

T: 

700 

= 

IO  - 

h  60 

or 

4 

+ 

24 

or 

2  H 

h  12 

i: 

750 

= 

IO  J 

h  65 

or 

4 

+ 

26 

or 

2  H 

-  13 

T  : 

800 

= 

10  - 

h  70 

or 

4 

+ 

28 

or 

2  H 

-  i4 

i: 

850 

= 

IO  - 

h  75 

or 

4 

+ 

30 

or 

2  H 

r  15 

i: 

900 

= 

10  - 

h  80 

or 

4 

+ 

32 

or 

2  H 

h  16 

i  : 

900 

= 

5  - 

h  40 

or 

4 

+ 

32 

or 

2  H 

hi6 

i: 

IOOO 

= 

5  - 

h  45 

or 

4 

+ 

36 

or 

2  - 

hi8 

i: 

I  IOO 

= 

5  - 

H  So 

or 

4 

+ 

40 

or 

2  - 

h  20 

i: 

I2OO 

= 

5  - 

H  55 

or 

4 

+• 

44 

or 

2  - 

h  22 

i: 

1300 

= 

5  - 

h  60 

or 

4 

+ 

48 

or 

2  - 

h  24 

i: 

I4OO 

= 

5  - 

r-  65 

or 

4 

+ 

52 

or 

2  - 

h  26 

i: 

1500 

= 

5  - 

|-  70 

or 

4 

+ 

56 

or 

2  J 

h  28 

i  : 

l6oO 

= 

5  - 

1-  75 

or 

4 

+ 

60 

or 

2  - 

h3Q 

i: 

I7OO 

= 

5  - 

f-  So 

or 

4 

+ 

64 

or 

2  - 

h32 

i  : 

I800 

= 

5  - 

r-  85 

or 

4 

+ 

68 

or 

2  - 

H34 

i: 

I800 

= 

5  - 

f-  85 

or 

4 

+ 

68 

or 

2  - 

h  34 

i: 

2OOO 

= 

5  - 

f-  95 

or 

4 

+ 

76 

or 

2  - 

H38 

i  : 

2200 

= 

5  - 

f-  105 

or 

4 

+ 

84 

or 

2  - 

h  42 

i: 

2400 

= 

5  - 

f-  115 

or 

4 

+ 

92 

or 

2  - 

h46 

i: 

26OO 

= 

5  - 

f  125 

or 

4 

+ 

IOO 

or 

2  - 

H  50 

i: 

2800 

= 

5  - 

f  135 

or 

4 

+ 

1  08 

or 

2  - 

h  54 

i: 

3000 

= 

5  - 

f  145 

or 

4 

+ 

116 

or 

2  - 

h58 

1:3200   = 

5  - 

f  155 

or 

4 

+ 

124 

or 

2  - 

h  62 

IQ4  MEDICAL  BACTERIOLOGY 

SEEDING  TUBES 

The  seeding  tubes  are  glass  test-tubes  i  inch  in  diameter  and  about  3  inches 
long,  with  round  bottoms.  In  order  to  measure  the  disinfectant  into  them  they 
are  placed  in  a  suitable  wooden  stand  to  receive  them.  We  found  it  convenient 
to  use  a  wooden  block  containing  six  rows  of  15  holes  each  for  the  disinfectant 
to  be  tested  and  a  separate  stand  for  the  phenol  controls.  The  tubes  are 
placed  in  the  stand  and  each  marked  with  the  strength  of  dilution  it  is  to 
contain. 

Starting  with  the  lowest  dilution  (i.e.,  the  strongest),  the  cylinder  is  shaken, 
then  5  cc.  are  measured  into  the  tubes  marked  to  receive  that  strength,  using 
a  5-cc.  delivery  pipette.  In  order  to  economize  glassware,  the  same  pipette  is 
used  for  measuring  out  the  next  dilution,  first  blowing  out  as  much  of  the  re- 
maining liquid  as  possible;  then  drawing  a  pipetteful  of  the  next  dilution  to  be 
used  and  discarding  that;  then  filling  the  pipette  a  second  time,  which  is  emptied 
into  the  seeding  tube. 

The  measuring-out  being  completed,  the  tubes  are  placed  in  the  water  bath 
and  allowed  to  stand  a  few  minutes  in  order  that  the  disinfectant  solution  may 
reach  the  standard  temperature.  We  have  not  found  it  necessary  to  use  cotton 
plugs  in  the  seeding  tubes.  They  are  sterilized  in  paper-lined  wire  baskets, 
with  the  closed  end  of  the  tubes  up. 

SUBCULTURE  TUBE  RACKS 

Wooden  racks,  with  five  rows  of  14  holes  each,  are  used  for  holding  the  sub- 
culture tubes,  and  as  plants  are  made  from  each  mixture  of  culture  and  disin- 
fectant every  2^  minutes  up  to  15  minutes,  six  tubes  are  required  for  each 
dilution.  Thus,  in  each  rack  we  have  ten  rows  of  six  tubes  each  with  two 
empty  cross  rows  of  holes  remaining,  which  are  utilized  by  placing  over  in  the 
next  row  each  tube  as  it  is  planted.  This  makes  it  easy  to  keep  run  of  the  tubes 
that  are  planted.  It  is  well  also  always  to  plant  from  the  seeding  tube  in  a 
certain  hole  in  the  water  bath  into  a  certain  row  of  tubes  in  the  rack.  This, 
after  a  little  practice,  will  help  to  avoid  errors  in  planting. 


METHOD  OF  CONDUCTING  THE  TEST 

If  there  are  in  one  experiment  more  than  10  dilutions  of  the  disinfectant, 
including  the  phenol  controls,  the  stronger  solutions  of  the  disinfectant  and 
phenol  are  tested  first,  as  it  will  not  be  necessary  to  plant  them  after  7^  min- 
utes. The  weaker  solutions  are  then  immediately  done  and  are  then  planted 
every  2%  minutes  for  15  minutes. 

For  keeping  the  time  a  stop-watch  can  be  used,  but  an  ordinary  watch  will 
serve  the  same  purpose  by  simply  starting  on  the  2^-  or  5-minute  periods. 


GERMICIDAL   POWER   OF    CHEMICAL   DISINFECTANTS  195 

When  everything  is  in  readiness  the  culture  is  added  to  the  disinfectant  solu- 
tions with  a  sterile  pipette  in  quantities  of  Ko  cc-  to  each  dilution. 

To  add  the  culture,  the  seeding  tube  containing  the  disinfectant  is  removed 
from  the  water  bath  with  the  left  hand  and  slanted  at  an  angle  of  about  45°, 
and  with  the  right  hand  the  end  of  the  pipette  containing  the  culture  is  intro- 
duced and  lightly  touched  against  the  side  of  the  tube  where  the  liquid  has  run 
away  on  account  of  the  slanting.  At  the  proper  time  the  culture  is  allowed  to 
run  into  the  disinfectant  solution,  the  pipette  removed,  the  tube  straightened 
up,  gently  shaken  three  times,  and  replaced  in  the  water  bath.  The  other 
tubes  are  done  the  same  way  in  succession,  and  it  will  be  found  that  15 
seconds  is  ample  time  for  each  tube.  By  adding  the  culture  to  the 
disinfectant  with  a  pipette  touched  against  the  side  of  the  seeding  tube, 
accurate  measurements  can  be  made  and  each  tube  receive  exactly  the 
same  amount  of  "seeding,"  which  is  not  the  case  when  the  culture  is  added 
by  the  "  drop." 

If  10  tubes  are  to  be  inoculated,  only  a  few  seconds  will  remain  after 
inoculating  the  last  tube  before  a  plant  from  the  first  tube  will  have  to 
be  made. 

The  mixing  tubes  are  not  removed  or  disturbed  in  making  the  planting  except 
to  insert  the  loop  or  spoon  into  them,  touch  the  bottom,  withdraw,  and  then 
make  the  plant  in  broth.  Every  effort  is  made  to  insert  and  withdraw  the  loops 
or  spoons  in  a  uniform  manner.  The  loops  and  spoons  are  bent  to  an  angle  of 
about  45°,  where  they  are  joined  to  the  shank,  and  therefore  are  always  filled 
with  the  mixture  when  withdrawn  from  the  seeding  tubes.  After  making  the 
plants  the  loops  or  spoons  are  flamed  as  already  described. 

After  an  experiment  is  finished  the  date  and  any  necessary  details  can  be 
marked  on  one  of  the  broth  tubes  and  the  rack  placed  in  the  incubator  at  37°C. 
for  48  hours.  At  the  end  of  this  time  the  results  are  recorded  on  a  chart  specially 
devised  for  the  purpose. 

DETERMINING  THE  COEFFICIENT 

After  a  large  number  of  experiments  we  have  concluded  that  the  method 
employed  by  the  Lancet  commission,  with  certain  modifications,  is  the  best  one 
for  determining  the  coefficient — i.e.,  the  mean  between  the  strength  and  time 
coefficients. 

In  performing  the  test,  plants  are  made  every  2%  minutes  up  to  and  includ- 
ing 15  minutes.  To  determine  the  coefficient,  the  figure  representing  the  degree 
of  dilution  of  the  weakest  strength  of  the  disinfectant  that  kills  within  2%  min- 
utes is  divided  by  the  figure  representing  the  degree  of  dilution  of  the  weakest 
strength  of  the  phenol  control  that  kills  within  the  same  time.  The  same  is 
done  for  the  weakest  strength  that  kills  in  15  minutes.  The  mean  of  the  two 
is  the  coefficient.  The  method  of  determining  the  coefficient  will  be  seen  in 
Table  17. 


Ip6  MEDICAL  BACTERIOLOGY 

TABLE  17 
Name,  "A." 

Temperature  of  medication,  2o°C. 

Culture  used,  B.  typhosus,  24-hour,  extract  broth,  filtered. 
Proportion  of  culture  and  disinfectant,  o.i  cc.  +  5  cc. 


Sample                    Dilution 
Phenol.                                  :  80 

T 
+ 

ime  culture  exposed  to  action  of 
disinfectant  for  minutes 

Phenol  coefficient 

5 

7* 

IO 

,,M 

IS 

- 

— 

- 

ff§ 

:QO 
:ioo 

:  no 

+ 

+ 

+ 

+ 

+ 

— 

to 

+ 

Disinfectant  "  A  "  1350 
:37.S 

— 

— 



| 

H       ON 
M      Ca 
0      0 

1400 

+ 

— 

— 

•     — 

4^ 

=  425 

+ 

+ 

— 

— 



— 

* 

:5oo 
'550 

J 

J 

~ 

- 

- 

- 

2 

:6oo 
1650 
1:700 

+ 

+ 

+ 

_j_ 

i 

" 

II 
tn 

% 

1:750 

+ 

+ 

! 

B.     The  Determination  of  the  Coefficient  with  the  Addition  of  Organic  Matter 

'It  may  be  well  to  briefly  discuss  here  the  meaning  or  significance  of  the  term 
"phenol  coefficient/'  particularly  when  it  is  determined  in  the  presence  of 
organic  matter.  In  general  terms  the  coefficient  of  a  disinfectant  may,  for 
practical  purposes,  be  defined  as  the  figure  that  represents  the  ratio  of  the 
germicidal  power  of  the  disinfectant  to  the  germicidal  power  of  carbolic  acid, 
both  having  been  tested  under  the  same  conditions. 

Although  the  germicidal  power  of  carbolic  acid  is  taken  as  the  unit  of  com- 
parison, it  is  influenced  to  a  certain  extent  by  conditions,  particularly  the  addi- 
tion of  organic  matter,  or,  in  other  words,  it  is  not  a  constant  unit.  This  has 
to  be  borne  in  mind  when  making  a  comparison  of  the  relative  values  of  the 
phenol  coefficients  of  a  disinfectant  determined  with  and  without  the  addition 
of  organic  matter,  respectively.  It  will  readily  be  seen,  for  instance,  that  if  the 
germicidal  powers  of  a  disinfectant  and  of  carbolic  acid  were  proportionately 
reduced  by  the  addition  of  organic  matter  the  coefficient  of  the  disinfectant 
would  remain  unchanged,  regardless  of  whether  or  not  organic  matter  was  used. 
However,  the  germicidal  power  of  carbolic  acid,  like  the  other  pure  phenols,  is, 
as  compared  with  most  other  disinfectants,  only  slightly  affected  by  the  addition 
of  organic  matter,  and  therefore  serves  as  a  fairly  accurate  means  of  estimating, 
in  the  presence  of  organic  matter,  the  germicidal  values  of  disinfectants  in 
general. 

The  method  of  determining  the  coefficient  of  disinfectants,  with  the  addition 
or  organic  matter,  is  identical  in  many  respects  with  the  method  in  which  no 


GERMICIDAL   POWER    OF    CHEMICAL   DISINFECTANTS 


IQ7 


organic  matter  is  used,  the  former  differing  from  the  latter  principally  in  the 
strengths  of  the  disinfectant  dilutions  that  have  to  be  prepared  and  in  the 
preparation  and  addition  of  the  organic  matter  to  the  disinfectant  dilutions 
when  performing  the  test.  As  the  method  in  which  no  organic  matter  is 
added  has  already  been  given  in  detail,  further  description  here  will  only 
consist  of  the  differences  between  the  two  methods. 

DILUTIONS 

In  making  the  dilutions  of  a  disinfectant  for  determining  its  coefficient  in  the 
presence  of  organic  matter,  allowance  must  be  made  for  the  further  dilution  of 
the  disinfectant  when  the  volume  of  organic  matter  is  added  thereto.  For 
instance,  if  i  cc.  of  organic  matter  is  added  to  5  cc.  of  a  i  per  cent,  dilution  of  a 
disinfectant  the  percentage  strength  of  the  disinfectant  is  proportionately 
reduced,  it  then  being  about  0.83  per  cent. 

The  volume  of  organic  matter  that  can  be  added  to  the  disinfectant  dilu- 
tion is,  of  course,  variable,  as  is  also  the  volume  of  disinfectant  dilution  to  which 
it  can  be  added.  Consequently,  we  have  decided  rather  arbitrarily  to  use  the 
organic  matter  by  adding  i  cc.  of  it  to  4  cc.  of  the  disinfectant  dilution  con- 
tained in  a  seeding  tube.  It  will  be  seen  that  the  strength  of  the  disinfectant  is 
reduced  20  per  cent,  by  the  addition  of  the  organic  matter  and  that  the  dilutions 
of  the  disinfectant  must  be  made  accordingly.  For  example,  if  it  is  desired  to 
test  a  i  per  cent,  strength  of  a  disinfectant  it  is  necessary  to  prepare  a  strength 
of  1.25  per  cent.,  4  cc.  of  which  becomes  a  i  per  cent,  strength  when  i  cc.  of  the 
organic  matter  is  added  to  it. 

We  also  tried  adding  2.5  cc.  of  the  organic  matter  to  2.5  cc.  of  the  disinfect- 
ant dilution,  but  found  it  rather  difficult  and  cumbersome  to  do,  particularly 
when  the  experiment  has  to  be  performed  with  a  number  of  different  strengths  of 
the  disinfectant.  When  using  the  proportions  as  just  stated,  the  dilutions  of  the 
disinfectant  are  made  double  the  strengths  it  is  desired  to  test,  thus  allowing  for 
the  further  dilution  when  an  equal  volume  of  organic  matter  is  added  thereto. 

In  using  the  proportions  of  i  cc.  of  organic  matter  to  4  cc.  of  the  disinfectant 
dilution  we  have  found  the  following  tables  (18  and  19)  of  service  in  preparing 
the  dilutions  of  the  disinfectant. 

TABLE  18.— STOCK  5  PER  CENT.  SOLUTION 
[5  cc.  of  disinfectant  -f-  95  cc.  distilled  water  =  solution  A] 


Strength 
to  be  tested 


•35 

:4o 


Strength 
to  be  made 


of 


:4o 
144 
:48 


1:56 


c. 
A 

Cc.  of 
dist.  water 

Cc. 

of  A 

Cc.  c 

dist.  w; 

20    + 

4 

or 

10 

+            2 

20    + 

8 

or 

10 

+          4 

20    + 

12 

or 

IO 

+         6 

20    + 

16 

or 

IO 

+         8 

20    + 

20 

or 

10 

+           IO 

20    + 

24 

or 

IO 

+          12 

20    + 

28 

or 

IO 

+       14 

2O    + 

32 

or 

IO 

+       16 

2O    + 

36 

or 

IO 

+       18 

10    + 

18 

or 

5 

+          9 

198 


MEDICAL  BACTERIOLOGY 


TABLE  18. — STOCK  5  PER  CENT.  SOLUTION. — (Continued} 


Strength 
to  be  tested 

Strength 
to  be  made 

Cc.             Cc.  of                     Cc.             Cc.  of 
of  A          dist.  water                    of  A          dist.  water 

1:80 

1:64 

=         10    + 

22 

or 

5  +       ii 

1:90 

1:72 

=         10    + 

26 

or 

5  +       i3 

1:100 

1:80 

=         10    + 

30 

or 

5  +       *5 

i:  no 

1:88 

=         10    + 

34 

or 

5  +       17 

i:  1  20 

1:96 

=         10    + 

38 

01 

5  +       T9 

1:130 

i:  104 

=         10    + 

42 

01 

5  +       21 

1:140 

1:112 

=        10    + 

46 

or 

5  +       23 

1:150 

1:120 

=         10    + 

50 

or 

5  +       25 

i:  160 

1:128 

=        10    + 

54 

or 

5  +       27 

i:  160 

1:128 

=         10    + 

54 

or 

5  +       27 

1:180 

1:144 

=         10    + 

62 

or 

5  +       31 

i:  200 

i:  160 

=         10    + 

70 

01 

5  +       35 

1:200 

i:  160 

=        5  + 

35 

or 

2    +           14 

1:225 

1:180 

=        5  + 

40 

or 

2    +          16 

1:250 

i:  200 

=        5  + 

45 

01 

2    +          18 

1:275 

i:  220 

=        5  + 

50 

or 

2    +          20 

1:300 

1:240 

=        5  + 

55 

OI 

2    +          22 

1:325 

i:  260 

=        5  + 

60 

or 

2    +          24 

1:350 

1:280 

=        5  + 

65 

or 

2    +          26 

i:375 

1:300 

=        5  + 

70 

or 

2    +          28 

1:400 

1:320 

=        5  + 

75 

or 

2    +          30 

1:400 

1:320 

=        5  + 

75 

or 

2    +          30 

7:450 

1:360 

=        5  + 

80 

or 

2    +          32 

i  :  500 

1:400 

=        5  + 

85 

or 

2  +       34 

1:550 

1:440 

5  + 

90 

or 

2    +          36 

1:600 

1:480 

=        5  + 

95 

or 

2    +          38 

1:650 

1:520 

=        5  + 

IOO 

or 

2    +          40 

TABLE  19.  —  STOCK  i  PER  CENT. 

SOLUTION 

[i  cc. 

disinfectant  + 

99  cc.  distilled 

water  =  solution  B] 

Strength  to  be 
tested 

Strength  to  be 
made 

Cc.           Cc.  of 
of  B     dist.  water 

1:500 

1:400 

5  +  15 

i:550 

1:440 

5  +  17 

1:600 

1:480 

5  +  19 

1:650 

1:520 

5  +  21 

i:  700 

1:560 

5  +  23 

1:750 

1:600 

5  +  25 

1:800 

1:600 

5  +  27 

1:850 

1:680 

5  +  29 

1:900 

i:  720 

5+3i 

1:900 

i:  720 

• 

5+3i 

i  :  1000 

1:800 

5+35 

i:  noo 

1:880 

5+39 

i  :  i  200 

1:960 

5+43 

1:1300 

i  :  1040 

5+47 

i:  1400 

i:  1120 

5  +  51 

1:1500 

i:  i  200 

5  +  55 

i:  1600 

i:  1280 

5  +  59 

i  :  i  700 

1:1360 

5+63 

i  :  1800 

1:1440 

5  +  67 

GERMICIDAL   POWER    OF    CHEMICAL   DISINFECTANTS  1 99 

It  will  be  seen  that  by  preparing  the  dilutions  of  the  disinfectant  according 
to  the  strengths  shown  in  the  second  column  of  Table  18,  and  then  adding  i  cc. 
of  organic  matter  to  4  cc.  of  the  dilutions,  the  final  dilutions  of  the  disinfectant 
become  as  represented  in  the  first  column  of  the  table  given  above. 

PREPARATION  AND  USE  OF  THE  PEPTONE -GELATIN  ORGANIC  MATTER 

As  already  stated,  we  prefer  to  use  a  mixture  of  peptone  and  gelatin  dissolved 
in  distilled  water.  It  is  prepared  from  Witte's  petpone  (siccum)  and  "Best 
French  Gold  Label"  gelatin.  The  stock  preparation  is  made  to  contain  10  per 
cent,  peptone  and  5  per  cent,  gelatin.  Proportionate  amounts  of  peptone  and 
gelatin  respectively,  are  weighed  out  and  liquefied  separately  in  small  quantities 
of  water  by  means  of  heat.  They  are  then  mixed  in  a  graduate  and  sufficient 
water  added  thereto  to  make  a  mixture  containing  10  per  cent,  of  peptone  and  5 
per  cent,  of  gelatin.  It  is  then  placed  in  bottles  of  appropriate  size  and  sterilized 
on  3  successive  days.  When  the  mixture  has  become  cold  it  will  be  observed 
that  some  of  the  peptone  settles  to  the  bottom  of  the  bottles  as  a  flocculent 
deposit.  Consequently,  the  bottle  should  be  shaken  before  using  it.  The 
stock  preparation  containing  5  per  cent,  gelatin  becomes  semi-solid  if  it  is  kept 
in  the  cold  room  at  a  temperature  of  i6°C.  However,  by  warming  it  until  it  be- 
comes perfectly  liquefied  and  then  not  allowing  it  to  go  below  the  temperature  of 
2O°C.  we  found  that  it  remains  liquid  and  is  easily  measured  in  the  pipette. 

By  adding  i  cc.  of  the  stock  preparation  of  10  per  cent,  peptone  and  5  per 
cent,  gelatin  to  4  cc.  of  the  disinfectant  dilutions  a  resulting  mixture  is  obtained- 
containing  2  per  cent,  of  peptone  and  i  per  cent,  of  gelatin,  or  a  total  of  3  per 
cent,  of  organic  matter. 

THE  METHOD  OF  CONDUCTING  THE  TEST 

Four  cc.  of  each  disinfectant  dilution,  including  the  phenol  controls,  are  ac- 
curately measured  into  the  seeding  tubes  and  placed  in  the  water  bath.  For 
reasons  that  will  be  obvious  later,  it  is  difficult  to  handle  more  than  nine  dilu- 
tions at  one  time  in  making  a  test.  The  broth  culture  of  B.  typhosus  is  filtered 
and  placed  in  the  water  bath.  The  desired  quantity  of  stock  peptone-gelatin 
mixture  is  measured  into  a  large  test-tube  or  flask  and  also  placed  in  the  water 
bath.  It  is  necessary  to  have  a  slight  excess  of  the  organic  matter,  so  that  there 
will  be  no  trouble  in  getting  the  ic-cc.  pipette  full  when  adding  the  organic 
matter  and  culture  to  the  disinfectant.  Thus,  for  nine  tubes  we  usually  meas- 
ure out  15  cc.  of  the  organic  matter  When  the  disinfectant  dilutions,  the  ty- 
phoid culture,  and  the  organic  matter  have  reached  the  temperature  of  the  water 
bath  (20°C.)  1.5  cc.  of  the  typhoid  culture  is  added  to  the  15  cc.  of  organic 
matter  and  thoroughly  mixed  by  means  of  a  zo-cc.  pipette.  With  the  same 
pipette  the  seeding  tubes  containing  the  disinfectant  dilutions  then  have  added 
to  them,  successively  every  15  seconds,  i.i  cc.  of  the  mixture  of  the  organic 
matter  and  typhoid  culture.  The  technique  of  shaking,  planting,  etc.,  is  then 
as  has  already  been  described. 


200  MEDICAL  BACTERIOLOGY 

From  the  above  it  will  be  seen  that  each  seeding  tube,  as  the  test  is  con- 
ducted, contains  4  cc.  of  the  disinfectant  dilution  plus  i  cc.  of  organic  matter, 
plus  o.i  cc.  of  typhoid  culture,  and  that  the  quantity  of  organic  matter  repre- 
sented is  3  per  cent. 

In  computing  the  strengths  of  the  disinfectant  and  the  percentage  of  organic 
matter  present  in  the  seeding  tubes  when  the  test  is  made,  we  have  disregarded 
the  slight  change  resulting  from  the  addition  of  the  o.i  cc.  of  typhoid  culture 
to  each  tube.  By  so  doing  the  experiment  is  very  much  simplified,  as  it  would  be 
very  difficult  to  prepare  the  dilutions  of  the  disinfectant  on  the  basis  of  allowing 
for  so  slight  a  change  as  is  caused  by  the  addition  of  o.i  cc.  of  liquid. 


CHAPTER  V 
DIAGNOSIS 

A  bare  knowledge  of  the  morphology  and  biology  of  pathogenic  bacteria  is  of 
little  value  in  diagnosis  unless  we  also  know  how,  when  and  where  to  search  for  a 
them  in  disease.  Some  of  this  information  has  been  imparted  in  the  previous 
chapters,  here  an  attempt  will  be  made  to  add  additional  information  that  may 
serve  in  attempts  to  disclose  the  offending  organism  present  in  infectious 
diseases. 

DISEASES  OF  THE  SKIN 

Some  skin  diseases  are  caused  by  bacteria  and  others  are  not.  Of  the  organ- 
isms which  attack  the  skin  the  staphylococcus  is  probably  the  most  frequent 
offender.  Since  the  staphylococcus  albus  is  practically  always  present  upon  the 
skin  in  health  and  disease,  we  cannot  with  assurance  look  upon  it  as  an  etiolog- 
ical  factor  when  obtained  in  smears  or  cultures  taken  from  skin  lesions,  except 
under  the  following  conditions: 

1.  When  possible  contamination  by  contact  with  the  surface  of  the  skin  of 
the  operator  or  of  the  patient  has  been  precluded. 

2.  When  the  organism  has  been  found  in  pure  culture  after  attempts  to  dis- 
cover other  organisms,  especially  the  acne  bacillus,  have  failed. 

ACNE 

Acne  may  be  caused  by  the  acne  bacillus  or  the  staphylococcus.  The 
staphylococcus  is  found  in  slides  and  aerobic  cultures  made  from  the  core  of  acne 
lesions  in  practically  all  cases,  whether  due  to  the  staphylococcus  or  the  acne 
bacillus. 

The  acne  bacillus  is  an  obligate  anaerobe,  to  obtain  it  in  pure  culture  from  an 
acne  lesion  proceed  as  follows:  Wash  the  skin  surrounding  the  pimple  with  soap 
and  water  and  alcohol,  remove  the  top  of  the  pimple  with  a  sterile  needle,  obtain 
the  lower  half  of  the  core  with  a  sterile  platinum  loop  (avoiding  contact  of  either 
loop  or  removed  material  with  the  patient's  skin  or  any  object),  plant  slant  tubes 
of  agar,  blood  serum  and  a  tube  of  bouillon  and  incubate  anaerobically  at  37°C. 
for  10  days  (by  incubating  for  10  days  the  acne  bacillus  will  outgrow  staphylo- 
cocci  that  are  present) .  After  planting  culture  media  obtain  a  core  as  before  and 
smear  in  a  thin  film  on  cover  glasses,  gently  heat  until  dry  and  stain  one  with 
methylene  blue,  another  by  Gram's  method.  The  acne  bacillus  is  somewhat 
similar  in  morphology  to  the  diphtheria  bacillus  and  is  Gram  positive. 

201 


202  MEDICAL  BACTERIOLOGY 

FURUNCULOSIS  AND  CARBUNCLES 

Furunculosis  and  carbuncles  are  commonly  caused  by  staphylococci,  some- 
times by  streptococci,  rarely  by  other  organisms.  To  make  a  (bacteriological 
diagnosis  obtain  material  as  when  examining  pimples,  plant  slant  tubes  of  agar 
and  blood  serum  and  incubate  aerobically  at  37°C.  for  24  to  48  hours.  Also 
make  slides,  stain  with  methylene  blue  and  by  Gram's  method. 

PHLEGMONA  D1FFUSA 

After  cleaning  the  surface  where  the  superficial  skin  is  unbroken,  and  over- 
lying pus  or  serum  exudate,  incise,  remove  several  loops  full  of  pus  or  serum  and 
plant  on  agar  and  blood  serum  and  incubate  at  37°C.  for  24  to  48  hours.  Make 
slides  for  microscopic  examination. 

ERYSIPELAS 

The  streptococcus  is  looked  upon  as  the  sole  cause  of  erysipelas,  not  every 
attempt  to  obtain  it  from  the  skin  lesions  succeeds.  Clean  the  surface  and 
attempt  to  withdraw  fluid  from  the  deep  layer  of  the  skin  and  subcutaneous 
tissues.  Plant  it  on  agar  and  blood  serum  and  incubate  at  37°C.  for  24  hours. 
Make  slides. 

Search  for  enlarged  glands  adjacent  to  the  diseased  area,  if  found,  massage, 
clean  overlying  skin  aspirate  some  of  the  contents  with  a  syringe.  Culture 
and  make  slides  from  material  so  obtained. 

MALIGNANT  PUSTULE 

Malignant  pustule  is  caused  by  the  anthrax  bacillus.  Clean  the  surrounding 
skin  and  overlying  tissue,  incise  and  obtain  some  serum.  Plant  on  agar  and 
blood  serum  and  make  slides  for  microscopic  examination. 

Withdraw  urine  with  catheter,  centrifuge  and  make  cultures  and  slides  from 
the  sediment. 

Withdraw  2  cc.  of  blood  from  a  vein  with  a  sterile  syringe,  smear  several 
drops  over  the  surface  of  each  of  several  tubes  of  agar  and  blood  serum  and  in- 
cubate at  37°C.  for  24  to  72  hours.  Make  smears  from  the  blood  and  stain  for 
microscopic  examination.  The  anthrax  bacillus  will  only  be  found  in  the  blood 
and  urine  when  the  disease  has  passed  beyond  the  initial  or  local  lesion  on  the 
skin  and  has  entered  the  circulation. 

FARCY  (GLANDERS) 

Glanders,  or  Farcy,  as  the  superficial  form  of  the  disease  is  known,  is  caused 
by  the  bacillus  mallei,  other  organisms,  as  staphylococci  frequently  contaminate 
superficial  lesions,  especially  if  open  and  exposed  to  the  air. 

Obtain  pus  or  scraping  from  a  lesion  and  plant  on  glycerin  agar,  blood  serum 
and  potato  and  incubate  aerobically  at  37°C.  for  24  to  72  hours,  also  make 
slides  and  stain. 


DIAGNOSIS  203 

PURPURA  HEMORRHAGICA 

Some,  probably  not  all  cases  of  this  disease,  are  of  bacterial  origin.  Cultures 
on  blood-agar,  ascitic  fluid  agarand  blood  serum  should  be  made  from  the  lesions, 
venous  blood  and  any  enlarged  glands  that  may  be  found. 

IMPETIGO  CONTAGIOSA 

Impetigo  contagiosa  may  be  caused  by  pathogenic  cocci.  Observing  the 
necessary  precautions  to  avoid  contamination  with  saprophytic  organisms 
present  upon  the  overlying  crusts  and  contiguous  skin,  cultures  should  be  made 
from  the  lesions  upon  agar  and  blood  serum. 

LUPUS 

Lupus,  tuberculosis  of  the  skin,  is  diagnosticated  bacteriologically  by  ex- 
amining scrapings  from  the  lesions  microscopically  for  tubercle  bacilli,  by  ex- 
cising a  portion  of  the  diseased  skin  and  after  proper  treatment  of  it  examining 
microscopically  both  for  tubercle  bacilli  and  typical  histological  changes,  by 
injecting  macerated  scrapings  from  the  lesions  into  guinea-pigs  and  rabbits  and 
by  subjecting  the  patient  to  the  tuberculin  test.  Not  all  these  procedures  are 
necessary  in  every  case.  The  multiplicity  of  methods  of  examination  is  de- 
manded because  one  or  several  may  fail  in  a  particular  case. 

FRAME  CES1A 

Frambcesia  or  yaws  is  a  non-veneral  skin  disease,  similar  to  some  of  the  super- 
ficial manifestations  of  syphilis.  It  is  caused  by  a  spirochaete  indistinguishable 
from  that  of  syphilis.  The  disease  is  diagnosticated  by  pinching  up  one  of 
the  lesions,  nicking  the  skin  with  a  sharp  knife  (avoid  producing  hemorrhage), 
and  collecting  the  serum  in  a  capillary  tube. 

The  serum  is  spread  in  a  thin  film  on  cover  glasses,  stained  according  to 
the  methods  given  for  treponema  pallidum,  and  examined.  Yaws  is  differen- 
tiated from  syphilis  microscopically  by  the  larger  number  of  spirochaete  found 
in  every  lesion  of  yaws  than  in  syphilis. 

SYPHILIS 

In  the  first  stage  of  syphilis  a  single,  indurated  ulcer  develops  at  the  site  of 
infection.  More  or  less  generalized  macular  and  papular  rashes  and  ulcers  occur 
from  time  to  time  during  the  second  stage  of  the  disease. 

The  treponema  may  be  found  in  the  eruptions  of  the  second  and  third  stages 
of  the  disease  by  following  the  technique  as  given  for  diagnosis  of  yaws.  It  is 
better,  however,  to  make  the  diagnosis  by  the  Wassermann  test. 

In  the  primary  stage  of  the  disease,  the  Wassermann  test  is  frequently  nega- 
tive, especially  early,  bacteriological  diagnosis  must  then  be  made  by  finding 
treponema  in  the  chancre  or  enlarged  glands.  After  the  chancre  has  been  free 
from  germicides  and  disinfectants  for  24  hours,  and  covered  with  a  sterile  dress- 
ing, the  dressing  is  removed,  the  ulcer  washed  with  sterile  salt  solution,  and 


204  MEDICAL  BACTERIOLOGY 

avoiding  the  production  of  hemorrhage,  serum  or  serum  and  scrapings  is  ob- 
tained from  the  floor  of  the  ulcer  with  a  capillary  tube.  If  adjacent  glands 
are  enlarged,  some  of  the  contents  of  these  is  removed  with  a  syringe.  This 
material  is  examined  unstained  with  the  dark-field  microscope,  or  stained,  as 
described  in  the  chapter  on  treponema  pallidum. 

MADURA  FOOT  AND  ACTINOMYCES 

In  these  infections  the  pus  is  searched  for  granules  and  these  are  crushed 
between  a  slide  and  a  cover  glass  and  examined  microscopically,  unstained  and 
after  staining.  When  granules  are  not  found,  the  pus  is  spread  on  slides  and 
examined  microscopically  before  and  after  staining. 

FAVUS,  RING  WORM,  PITYRIASIS  VERSICOLOR,  ERYTHRASMA  AND  BLASTOMY- 

CETIC  DERMATITIS 

Bacteriological  diagnosis  in  the  diseases  caused  by  the  higher  forms  of  bac- 
teria is  usually  based  upon  microscopic  examination  alone,  cultures  may  be 
made  for  more  accurate  classification.  In  the  above-named  diseases,  the 
method  of  procedure  is  the  same,  some  hairs,  portions  of  the  crust  or  scab  and 
scrapings  from  the  border  line  between  the  diseased  and  healthy  skin  should  be 
obtained  and  examined  separately.  A  number  of  slides  should  be  prepared 
and  examined  in  every  case.  The  material  to  be  examined,  whether  hair,  crust 
or  scrapings,  should  be  placed  in  a  drop  of  20  to  40  per  cent,  potassium  hydrate 
solution  and  a  cover  glass  dropped  upon  it.  The  slides  are  then  allowed  to 
stand  at  room  temperature  for  several  minutes.  Then  some  'are  examined 
without  heating,  others  after  gently  warming  for  several  minutes  and  others 
after  heating  until  steam  arises. 

In  every  case  where  the  microscopical  examination  and  other  findings  do 
not  disclose  the  exact  nature  of  the  offending  organisms,  cultures  should  be 
made. 

LEPROSY 

In  every  suspected  case  of  leprosy  the  anterior  nares  should  be  examined 
whether  lesions  appear  there  or  not;  a  sterile  cotton  swab  is  passed  over  the 
mucous  membrane  and  then  rubbed  on  a  slide.  Skin  ulcers  should  be  cleansed 
to  free  them  of  contamination  and  slides  made  from  scrapings  removed  from 
the  floor  of  the  ulcer. 

These  slides  should  be  stained  as  when  examining  for  tubercle  bacilli. 

DISEASES  OF  THE  EYE 

The  lids  are  subject  to  all  of  the  infectious  diseases  that  affect  the  skin  and 
the  technique  of  bacteriological  diagnosis  is  the  same  as  in  similar  diseases  of 
the  skin  of  other  parts  of  the  body. 

The  lachrymal  apparatus  is  more  frequently  attacked  by  the  pyogenic  or- 
ganisms than  by  other  bacteria.  When  a  bacteriological  diagnosis  of  an  infec- 
tion of  any  part  of  the  lachrymal  apparatus  is  desired,  the  affected  and  con- 


DIAGNOSIS  205 

tiguous  parts  should  be  thoroughly  washed  out  with  sterile  salt  solution  and 
covered  with  a  sterile  dressing.  About  12  hours  later  the  dressing  is  removed, 
a  sterile  loop  or  cotton  swab  is  passed  over  the  affected  membrane  and  then 
drawn  over  the  surface  of  slant  tubes  of  agar  and  blood-serum  media  the  tubes 
are  incubated  at  37°C.  for  18  to  72  hours.  Slides  should  also  be  prepared. 

CONJUNCTIVA 

In  addition  to  acute  catarrhal,  contagious  conjunctivitis  or  pink-eye,  caused 
by  the  Koch- Weeks  bacillus  and  ophthalmia  neonatorum  caused  by  gonococcus, 
infections  of  the  conjunctiva  may  be  caused  by  a  variety  of  organisms.  After 
cleansing  and  covering  with  a  sterile  dressing  for  a  number  of  hours,  slides 
should  be  made  and  stained  with  methylene  blue  and  by  Gram's  method,  cul- 
tures should  be  made  on  appropriate  media.  The  material  for  examination  is 
obtained  by  passing  a  sterile  cotton  swab  over  the  affected  part. 

SYPHILIS  AND  TUBERCULOSIS  OF  THE  EYE 

Chancres,  though  rarely  observed  in  such  localities,  do  occur  on  the  lids  and 
conjunctiva,  suspicious  ulcers  should  be  examined  for  treponema  pallidum  and  a 
Wassermann  test  made. 

Secondary,  tertiary  and  congenital  syphilis  frequently  cause  inflammation 
of  the  cornea,  sclera,  iris,  ciliary  body,  choroid,  retina  and  optic  nerve,  occasion- 
ally tuberculosis  does  the  same. 

In  suspected  cases  a  Wassermann  test  should  be  made  and  if  negative  a 
tuberculin  test  is  made. 

DISEASES  OF  THE  EAR 

The  auricle  and  external  auditory  canal  are  subject  to  all  the  infections  that 
affect  the  skin,  but  they  are  much  less  frequently  attacked.  The  technique 
of  bacteriological  diagnosis  of  infections  of  those  parts  is  the  same  as  in  similar 
infections  of  the  skin. 

The  diphtheria  bacillus  sometimes  infects  the  external  auditory  canal,  this 
is  most  apt  to  occur  when  the  throat  is  also  involved  and  technique  for  examina- 
tion is  the  same  as  when  examining  the  throat  for  diphtheria  bacilli. 

OTITIS  MEDIA 

Nearly  all  infections  of  the  middle  ear  follow,  or  occur  as  extensions  of,  naso- 
pharyngeal  infections.  When  they  progress  to  suppuration  and  discharge 
through  the  external  auditory  canal  secondary  infection  from  the  air  frequently 
results. 

Bacteriological  diagnosis  is  usually  called  for  when  the  disease  has  progressed 
to  suppuration  and  pus  is  discharging  through  the  external  meatus.  In  such 
cases  the  ear  is  thoroughly  cleansed  by  syringing  with  sterile  salt  solution,  a 
plug  of  sterile  cotton  is  then  placed  in  the  external  meatus,  6  to  24  hours  later 
the  auricle  is  asepticized,  the  plug  removed,  and  a  sterile  cotton  swab  is  passed 
into  the  external  auditory  canal  to  collect  the  exudate.  Slides  and  cultures  on 


206  MEDICAL  BACTERIOLOGY 

agar,  ascitic  agar  and  blood  serum  are  made.  The  organisms  most  frequently 
found  as  the  cause  of  otitis  media  are  the  following:  staphylococci,  bacillus 
pyocyaneus,  streptococci,  pneumococcus,  bacillus  ozena,  diphtheria  bacillus  and 
bacillus  of  influenza. 

MASTOIDITIS 

Mastoid  infections  are  usually  the  result  of  extension  from  the  middle  ear. 
DISEASES  OF  THE  NOSE 

Primary,  secondary  and  tertiary  lesions  of  syphilis  occur  occasionally  in  the 
nose  and  are  diagnosticated  by  examination  of  lesions  for  treponema  pallidum 
and  chiefly  by  the  Wassermann  test. 

Diphtheria  bacilli  may  be  present  in  the  nose  and  not  elsewhere,  in  persons 
not  suffering  with  diphtheria  carriers.  Nasal  diphtheria  may  occur  without 
the  throat  being  involved;  this  is  rare. 

Chronic  rhinitis,  chronic  nasal  catarrh,  may  be  caused  by  staphylococci,  the 
pneumococcus,  bacillus  of  rhinoscleroma,  bacillus  of  ozena  and  less  frequently 
micrococcus  catarrhalis,  meningococcus  and  other  organisms.  Any  of  these 
organisms  may  be  present  at  times  upon  normal  nasal  mucosa. 

To  make  a  bacteriological  examination  pass  a  sterile  cotton  swab  into  each 
nostril,  brush  it  over  as  much  of  the  membrane  as  possible,  withdraw  and  make 
surface  plants  upon  agar,  ascitic  fluid  agar  and  blood  serum.  Also  prepare 
slides  for  microscopic  examination. 

When  possible  it  is  well  to  make  a  second  set  of  cultures  and  slides  after 
preliminary  treatment  of  the  nose  as  follows: 

Thoroughly  cleanse  the  nose,  wash  out  with  sterile  salt  solution  and  place  a 
plug  of  cotton  in  each  nostril  so  as  to  filter  the  air  without  impairing  inspiration, 
keep  the  patient  in  a  room  free  of  gas  for  12  hours  and  then  obtain  material  for 
culture  and  slides. 

DISEASES  OF  THE  LUNGS 

Bacteriological  diagnosis  of  diseases  of  the  lungs  (other  than  syphilis  which  is 
rare)  is  based  upon  the  microscopic  examination  of  the  sputum,  occasionally 
supplemented  by  cultures  from  sputum  and  inoculation  of  the  sputum  into 
rabbits  and  guinea-pigs. 

The  technique  for  obtaining  sputum  and  examining  it  has  already  been  con- 
sidered in  the  chapters  on  the  pneumococcus  and  tubercle  bacillus.  Here  it  is 
only  necessary  to  add  that  pneumonia  is  not  a  specific  disease.  While  the 
pneumococcus  is  responsible  for  most  cases  of  acute  pneumonia,  still,  a  large 
number  of  cases  are  caused  by  other  organisms,  staphylococci,  bacillus  of 
influenza,  streptococci  and  the  tubercle  bacillus.  In  acute  pneumonia,  caused 
by  the  pneumococcus,  that  organism  is  usually  found  to  predominate  in  the 
sputum  some  time  during  the  disease,  in  nearly  all  cases,  yet  it  must  be  remem- 
bered that  occasionally  it  does  not,  especially  early  in  the  disease. 

It  is  most  important  to  remember  there  is  a  group  of  cases  which  start  as, 


DIAGNOSIS  207 

run  the  typical  course  of,  and  are  clinically  diagnosticated  as  acute  lobar  pneu- 
monia caused  by  the  pneumococcus;  that  bacteriological  examination  of  the 
sputum  prior  to  crisis  shows  a  preponderance  of  pneumococci  in  most  cases  and 
seldom  shows  tubercle  bacilli;  that  after  crisis  recovery  does  not  follow,  cough 
and  expectoration  continues,  resolution  is  incomplete,  the  sputum  may  still 
show  a  preponderance  of  pneumococci,  but  usually  many  organisms  are  present, 
tubercle  bacilli  are  not  discovered  for  weeks  or  months ;  prior  to  their  discovery 
it  becomes  obvious  that  the  case  is  one  of  pulmonary  tuberculosis.  In  all  such 
cases  where  resolution  and  convalescence  are  retarded  following  crisis  a  tuber- 
culin test  should  be  made. 

(DISEASES  OF  THE  GASTRO-INTESTINAL  TRACT  AND  CONTINUOUS  FEVERS 

The  signs,  symptoms  and  clinical  course  of  typhoid  fever,  paratyphoid 
fever,  food-poisoning,  tuberculosis,  syphilis,  gonococcus,  staphylococcus  and 
streptococcus  infections  and  malaria  are  at  times  indistinguishable,  except  by 
careful  bacteriological  studies. 

In  typhoid,  paratyphoid,  colon  bacillus  and  other  infections  caused  by 
members  of  the  typhoid-colon  group,  during  the  first  week  of  the  disease  the 
offending  organism  can  always  be  isolated  from  the  blood  by  culture.  Later 
than  the  second  week  the  offending  organism  or  its  specific  agglutinin  can  be 
found  in  the  blood  in  nearly  all  cases.  These  tests  should  be  made  in  all  such 
cases,  and  when  negative,  foci  of  infection  with  pyogenic  cocci  should  be  sought 
for;  blood  cultures  for  staphylococci,  streptococci  and  pneumococci  should  be 
made.  If  these  fail  to  establish  the  diagnosis  the  Wassermann  and  possibly 
the  gonococcus  complement  fixation  tests  should  be  made. 

In  suspected  cases  of  cholera  and  bacillary  dysentery  the  feces  should  be 
examined  by  culture  for  the  offending  organism  and  the  blood  serum  for  specific 
agglutinin  and  lysin. 

Huston  and  McCloy,  The  Lancet,  vol.  ii,  No.  15,  Oct.  7,  1916,  have  reported 
the  isolation  of  an  organism,  called  "  enterococcus "  from  the  feces  of  patients 
with  a  cryptogenic  illness,  which  they  believe  is  due  to  this  organism.  They 
believe  the  "  enterococcus  "  is  not  of  the  normal  bacterial  flora  of  the  intestine 
and  suggest  the  advisability  of  attempts  to  isolate  possible  causative  organisms 
from  the  feces  in  all  cases  of  cryptogenic  gastro-intestinal  disease. 

DISEASES  OF  THE  GENITO -URINARY  TRACT 
ULCERS  OF  THE  EXTERNAL  GENITALIA 

Ulcers  of  the  external  genitalia  may  be  due  to  physical,  chemical  or  vital 
causes.  Any  of  the  organisms  that  produce  ulceration  of  the  skin  elsewhere  may 
do  so  here.  As  a  matter  of  fact,  nearly  all  the  ulcers  of  the  external  genital 
organs  that  are  brought  to  the  bacteriologist  for  diagnosis  are  chancres  or 
chancroids,  the  result  of  venery,  and  the  problem  is  to  discover  whether  they 
are  caused  by  the  treponema  pallidum  or  other  organisms. 

The  technique  of  examination  is  as  follows:  Thoroughly  cleanse  the  ulcer 


208  MEDICAL  BACTERIOLOGY 

and  surrounding  parts  and  cover  with  a  sterile  dressing,  without  any  germicide 
or  antiseptic;  after  this  dressing  has  been  on  for  12  to  24  hours  remove  it  and 
obtain  some  serum,  also  scrapings  from  the  floor  of  the  ulcer;  examine  these 
specimens  unstained,  microscopically,  using  the  dark-field  microscope;  if  trepo- 
nema  pallidum  is  observed  the  diagnosis  is  established;  if  not  observed,  proceed 
as  follows:  obtain  serum  and  scrapings  from  the  ulcer,  spread  on  slides  or  cover 
glasses,  dry  without  heating  and  stain — some  with  methylene  blue,  others  by 
Gram's  method  and  others  by  Giemsa's  method  for  treponema  pallidum;  if 
desired,  cultures  may  be  made  on  blood-agar,  ascitic  agar  and  inoculation  into  a 
rabbit's  testicle. 

The  treponema  pallidum  is  present  in  chancres,  either  in  pure  culture  or 
together  with  other  organisms. 

Chancre  and  chancroid  may  exist  together,  in  which  case  the  organisms  of 
both  conditions  will  be  found. 

Ducrey's  bacillus  (bacillus  of  soft  sore)  is  considered  the  specific  cause  of 
chancroid.  Other  organisms  are  usually  found  with  it — staphylococci,  gono- 
cocci  or  micrococcus  tetragenus. 

Ducrey's  bacillus  is  a  short  rod  or  oval,  0.5  fj,  by  2.0  /*,  has  round  ends,  is  non- 
motile,  is  arranged  singly  and  in  chains,  occurs  within  pus  cells  and  outside  of 
them,  stains  readily  with  all  the  usual  anilin  dyes  and  is  Gram  negative.  It  does 
not  stain  uniformly,  the  poles  stain  deep,  the  intervening  portion  faintly  or  not 
at  all. 

Ducrey's  bacillus  does  not  grow  on  ordinary  media.  On  blood-agar  in- 
cubated aerobically  at  3i°C.,  round,  raised,  pin-head-sized,  opaque,  grayish 
colonies  develop  in  24  to  48  hours. 

ACUTE  URETHRITIS  AND  VAG1NITIS 

Acute  urethritis  or  vaginitis  may  be  caused  by  staphylococci  or  gonococci, 
rarely  by  other  organisms.  Microscopic  examination  of  smears  made  from  the 
discharge  and  stained  by  methylene  blue  and  by  Gram's  method  will  establish 
the  diagnosis  in  nearly  all  cases. 

CHRONIC  URETHRITIS  AND  VAGINITIS 

Chronic  urethritis  and  vaginitis  may  be  due  to  a  single  organism,  usually 
several  are  present.  When  due  to  a  single  organism  it  may  be  impossible  to 
find  it  except  following  an  unusual  irritation  or  hypersecretion  such  as  may  be 
produced  by  drinking  beer,  massage  of  the  prostate  or  sexual  excitement.  If 
spreads  and  cultures  fail  to  show  the  offending  organism,  they  should  be  again 
made  after  instituting  irritation  or  hypersecretion.  Cultures  are  made  by 
picking  up  some  of  the  discharge  and  planting  it  on  the  surface  of  Loeffler's 
blood-serum  medium  and  on  neutral  ascitic  agar.  Tubes  containing  no  free 
moisture  must  be  used  and  it  is  best  to  plant  several  so  that  some  may  be  incu- 
bated with  reduced  oxygen  tension  and  others  aerobically  at  37°C. 

When  chronic  urethritis  or  vaginitis  is  due  to  several  organisms  there  is  usu- 
ally sufficient  discharge  to  permit  of  diagnosis  by  microscopic  examination  of 


DIAGNOSIS  209 

smears  and  by  culture  at  any  time.  In  such  cases  gonococci,  staphylococci, 
pseudodiphtheria  bacilli,  the  colon  bacillus  or  the  diphtheria  bacillus  may  be 
present. 

INFECTION  OF  THE  URINARY  BLADDER 

Bacteriologically,  infection  of  the  urinary  bladder  can  only  be  determined 
with  accuracy  by  microscopic  and  cultural  examination  of  urine  obtained 
through  a  sterile  catheter  under  conditions  which  preclude  the  possibility  of 
bacteria  entering  the  urine  outside  the  bladder. 

INFECTION  OF  THE  KIDNEYS 

Bacteriologically,  renal  infection  can  only  be  detected  by  microscopic  and 
cultural  examination  of  urine  obtained  through  ureteral  catheter. 

Any  of  the  organisms  that  may  infect  the  urethra  can  infect  the  bladder  or 
kidney.  The  colon  bacillus  is  probably  the  most  frequent  invader  of  the 
bladder  and  kidney. 

When  urine  is  to  be  examined  for  bacteria  other  than  tubercle  bacilli  the 
examination  should  be  made  immediately  after  removal  from  the  body.  The 
technique  described  on  pages  116  and  117  should  be  employed;  in  addition, 
plants  on  plain  agar,  or  better,  on  blood-agar,  should  also  be  made. 

For  the  tubercle  bacilli  see  page  98. 

There  is  a  wide  difference  of  opinion  as  to  the  significance  of  tubercle  bacilli 
found  in  urine,  also  as  to  whether  or  not  acid-fast  organisms  found  in  urine  may 
properly  be  considered  tubercle  bacilli. 

The  results  of  extensive  laboratory  studies  conducted  by  Prof.  R.  C.  Rosen- 
berger  and  the  coordinated  clinical  and  laboratory  findings  of  Dr.  Charles 
Bonney  and  the  writer  may  be  summarized  as  follows: 

First,  acid-fast  bacteria  (which  are  similar  to,  but  which  are  not  tubercle  or 
leprosy  bacilli,  such  as  occur  at  times  in  sputum,  smegma,  feces,  urine  and 
water),  occasionally  resist  decolorization  and  remain  red  after  immersion  in 
Pappenheim's  solution  for  2  minutes.  Rarely,  indeed,  less  than  once  in  a 
hundred  times,  do  such  organisms  remain  red  after  twenty  minutes'  immersion 
in  Pappenheim's  solution. 

Second,  we  have  never  found  red  bacilli  in  urine  from  clinically  non-tubercu- 
lous individuals  when  (a)  the  external  genital  organs  are  carefully  cleansed  prior 
to  micturition  or  catheterization,  (b)  the  urine  is  collected  in  sterile  containers 
and  protected  from  extra  corporeal  contamination,  and  (c)  the  smears  made  for 
microscopic  examination  are  immersed  in  Pappenheim's  solution  for  20  minutes 
or  more,  after  carbol  f uchsin  staining. 

Third,  whenever  we  have  found  red  bacilli,  identified  as  tubercle  bacilli,  in 
urine,  there  has  been  at  the  time,  or  later,  conclusive  evidence  of  active  tuber- 
culosis of  one  or  both  kidneys,  usually,  but  not  always,  associated  with  distinct 
physical  signs  of  latent  or  active  pulmonary  tuberculosis. 


2IO  MEDICAL  BACTERIOLOGY 

ARTHRITIS 

Most  cases  of  arthritis  are  due  to  infection.  It  is  generally  believed  that  the 
atria  of  infection  in  most  cases  is  through  the  tonsils  or  teeth.  When  tonsillar 
or  mouth  infection  and  arthritis  coexist,  the  cause  of  the  arthritis  is  sometimes 
sought  for  in  cultures  obtained  from  the  tonsils;  such  conclusions  are  quite  as  apt 
to  be  erroneous  as  correct. 

The  offending  organism  can  sometimes,  but  not  always,  be  found  in  fluid  or 
pus  aspirated  from  the  diseased  joint,  and  an  attempt  to  do  so  should  be  made 
when  bacteriological  diagnosis  is  attempted. 

In  nearly  all  cases  of  arthritis  caused  by  staphylococci,  streptococci  or  the 
pneumococcus,  careful  search  will  reveal  the  presence  of  enlarged  glands  adjacent 
to  the  affected  joint.  The  offending  organism  can  be  found  in  these  if  the  follow- 
ing procedure  is  carried  out  so  carefully  that  contamination  of  the  gland  with 
extraneous  bacteria  is  precluded: 

Asepticize  the  skin  overlying  the  enlarged  gland,  incise  the  skin  and  remove 
the  gland,  place  the  gland  in  a  sterile  mortar  and  emulsify  it  with  sterile  water, 
plant  on  slant  tubes  of  agar,  blood-agar  and  ascitic  fluid  agar,  also  make  deep 
stab  cultures  in  tubes  containing  agar  and  blood-agar  to  the  depth  of  6  inches. 
Incubate  aerobically  at  37°C. 

Inject  the  remaining  portion  of  gland  emulsion  into  the  peritoneal  cavity  of 
a  guinea-pig. 

It  is  to  be  remembered  that  a  large  number  of  "rheumatic"  manifestations, 
muscular  as  well  as  arthritic,  are  due  to  syphilis  and  that  the  Wassermann  test  is 
positive  in  75  per  cent,  or  more  of  these  syphilitic  cases. 

The  gonococcus  complement  fixation  test  will  often  establish  the  diagnosis, 
in  cases  of  gonococcus  arthritis,  when  other  means  fail. 

In  tubercular  arthritis  the  tubercle  bacillus  is  practically  never  found  in 
slides  or  cultures  made  from  the  fluid  or  pus;  animal  inoculations  sometimes  give 
positive  results.  In  suspected  cases  of  tubercular  arthritis,  when  other  means  of 
diagnosis  fail,  when  there  is  no  contraindication,  a  tuberculin  test  should 
be  made. 


CHAPTER  VI 
BACTERIAL  VACCINES 

Bacterial  vaccines  with  few  exceptions  as  noted  are  suspensions  or  solutions 
of  attenuated  or  dead  bacteria,  or  bacterial  products,  in  normal  salt  solution. 

Stock  vaccines  are  those  made  from  cultures  kept  in  the  laboratory. 

Autogenous  vaccines  are  those  made  from  cultures  of  the  offending  organism 
or  organisms  obtained  from  the  infected  person  and  used  in  the  treatment  of 
that  patient. 

Polyvalent  vaccines  are  those  made  from  various  strains  of  the  same  organ- 
ism obtained  from  different  sources.  Mixed  vaccines  are  those_containing  two 
or  more  different  species  of  bacteria. 

Some  vaccines  are  prepared,  standardized,  sterilized  and  administered  by 
special  methods.  In  general,  most  vaccines  are  made  from  24-hour-old  cultures 
on  the  surface  of  solid  media.  The  medium  used  depends  upon  the  require- 
ments of  the  organism  one  desires  to  make  the  vaccine  with.  Those  that  grow 
well  on  agar  are  planted  on  agar;  those  that  grow  best  on  blood-agar  or  ascitic 
agar  are  planted  on  them.  Young  cultures  are  most  desirable  but  if  growth  is 
not  apparent  in  24  hours  incubation  is  extended  to  48  or  occasionally  72  hours. 
The  bacteria  are  procured  by  pouring  sterile  normal  salt  solution  on  the  media 
and  shaking  or  scraping  until  the  bacteria  are  suspended  in  the  salt  solution. 

The  salt  solution  suspension  of  bacteria  is  then  poured  into  a  sterile  tube  or 
flask  and  shaken  to  break  up  clumps. 

The  number  of  bacteria  per  cubic  centimeter  is  estimated  by  mixing  equal- 
sized  drops  of  blood  and  bacterial  suspension  on  a  glass  slide,  spreading  in  a 
thin,  even  film,  staining  with  any  blood  stain  and  examining  under  an  oil  im- 
mersion lens.  A  satisfactory  count  can  only  be  made,  when  the  shaking  of  the 
bacterial  suspension  has  been  sufficient  to  break  up  clumps  and  evenly  distribute 
the  individual  organisms;  when  the  mixture  of  blood  and  bacterial  suspension 
has  been  done  so  rapidly  that  the  film  is  spread  before  clotting  occurs ;  when  the 
film  is  thin  enough  to  present  fields  containing  bacteria  and  red  cells  not  too 
numerous  to  count  when  examined  through  the  oil  immersion  lens.  The  bac- 
teria and  red  cells  are  counted  in  each  field  until  a  total  of  500  red  cells  has  been 
counted;  the  number  of  bacteria  counted  is  totaled.  Multiply  the  number  of 
bacteria  counted  by  10,000,000.  This  gives  the  number  of  bacteria  in  each 
cubic  centimeter  of  the  vaccine. 

This  computation  is  based  on  the  fact  that  normal  blood  contains  5,000,000,- 
ooo  red  cells  per  cubic  centimeter. 

After  the  number  of  bacteria  per  cubic  centimeter  has  been  determined  the 
suspension  of  bacteria  is  further  diluted  by  the  addition  of  sterile  normal  salt 
solution  so  as  to  bring  the  bacterial  content  to  1,000,000,000  per  cubic 
centimeter. 


212  MEDICAL  BACTERIOLOGY 

Vaccines  are  sterilized  by  heat  in  a  water  bath  at  the  minimum  temperature 
and  time  of  exposure  that  will  kill  the  organism. 

After  heating,  several  loopsful  of  the  vaccine  are  planted  on  culture  media 
and  incubated  to  prove  sterility. 

When  the  vaccine  has  been  sterilized,  o.io  to  0.25  per  cent,  of  tricresol  is 
added  and  the  vaccine  sealed  in  appropriate  sterile  containers. 

The  most  important  vaccines  made,  according  to  the  technique  just  de- 
scribed, will  be  mentioned,  also  the  media  upon  which  they  may  be  cultivated, 
temperatures  and  time  of  exposure  required  to  sterilize  and  purpose  for  which 
they  are  employed: 

TYPHOID  VACCINE 

All  strains  of  the  typhoid  bacillus  do  not  possess  equal  immunizing  proper- 
ties. One  having  proved  immunizing  power  is  used.  It  is  planted  on  plain 
agar.  The  bacterial  suspension  is  sterilized  in  a  water  bath  at  56°C.  to  6o°C.  for 
J^  hour. 

Typhoid  vaccine  is  used  to  produce  immunity,  to  combat  sequelae  of  typhoid 
fever  and  sometimes  in  the  treatment  of  typhoid  fever. 

Dose. — The  average  immunizing  dosage  consists  of  three  injections  at  in- 
tervals of  10  days;  the  first  500,000,000,  second  1,000,000,000  and  third  1,000,- 
000,000  organisms.  In  the  treatment  of  typhoid  fever  and  sequelae  the  dose 
varies  from  500,000  to  1,000,000,000  at  intervals  between  injections  from  2  to 
20  days. 

Vaccines  of  the  various  paratyphoid  bacilli,  the  bacillus  coli,  the  various 
dysentery  bacilli  and  the  spirillum  of  cholera  are  made  in  the  same  way,  have 
the  same  dosage  and  are  employed  principally  to  immunize  against  infection 
and  in  the  treatment  of  sequelae,  occasional  and  with  less  success  in  the  treat- 
ment of  subacute  or  prolonged  cases  of  these  diseases. 

GONOCOCCUS  VACCINE 

Gonococci  are  cultured  on  media  composed  of  about  6  parts  glycerin  agar 
and  4  parts  of  blood,  blood  serum,  ascitic  fluid,  or  egg.  The  vaccine  is  sterilized 
in  a  water  bath  at  55°C.  to  6o°C.  for  from  10  to  30  minutes.  Gonococcus 
vaccine  does  not  confer  immunity;  it  is  sometimes  of  value  in  acute  urethritis, 
but  seems  most"  effective  in  subacute  and  chronic  infections  and  sequelae  of 
gonorrhoea. 

The  average  initial  dose  for  children  is  5,000,000,  for  adults  25,000,000. 
Reactions  are  to  be  avoided. 

Meningococcus  vaccine  is  made  in  the  same  way  but  is  seldom  used  and 
there  is  not  much  evidence  in  regard  to  its  indications  or  value. 

STAPHYLOCOCCUS  VACCINE 

When  making  a  stock  vaccine  a  polyvalent  one  should  be  made.  If  it  is  to 
be  a  staphylococcus  aureus  vaccine,  then  pure  cultures  of  staphylococcus  aureus 
should  be  obtained  from  as  many  different  sources  as  possible — from  the  blood 


BACTERIAL  VACCINES  213 

^ 

in  cases  of  staphylococcus  aureus  bacteremia,  from  infected  wounds,  from 
abscesses,  from  the  throat  in  cases  of  staphylococcus,  tonsillitis,  etc. 

These  various  strains  are  cultivated  on  plain  agar,  suspended  in  salt  solution, 
mixed  and  sterilized  in  the  water  bath  at  6o°C.  to  8o°C.  from  30  minutes  to  i 
hour. 

Staphylococcus  vaccines  are  employed  to  fortify  natural  immunity  and  to 
combat  staphylococcus  infections.  The  average  initial  dose  is  from  50,000,000 
to  100,000,000. 

Staphylococcus  albus  and  citrus  vaccines  are  made  in  the  same  way;  the 
same  is  true  of  streptococcus  and  pneumococcus  except  that  these  two  are  best 
cultivated  on  blood-agar.  The  average  dose  of  all  is  the  same. 

Vaccines  of  any  of  these  cocci  seldom  produce  any  marked  exaltation  of 
immunity  when  given  to  healthy  persons;  they  do  not  as  a  rule  conspicuously 
modify  or  curtail  acute  infections,  their  chief  value  is  in  the  treatment  of  sub- 
acute  and  chronic  infections. 

There  are  innumerable  strains  of  staphylococci  that  are  indistinguishable 
but  differ  in  that  a  vaccine  made  from  one  does  not  stimulate  antibody  forma- 
tion or  immunity  to  the  others.  The  same  is  true  of  streptococci  and  pneumo- 
cocci.  Therefore,  autogenous  vaccines  are  very  much  superior  to  stock  vac- 
cines in  the  treatment  of  such  infections. 

SENSITIZED  VACCINES 

Sensitized  vaccines  are  prepared  by  cultivating  the  organism  on  its  appro- 
priate medium,  washing  off  the  growth  with  sterile  salt  solution,  shaking  the 
suspension  to  break  up  clumps  and  counting  the  number  of  bacteria  per  cubic 
centimeter  as  previously  described.  The  suspension  is  then  centrifugalized  at 
high  speed  and  when  the  bacteria  have  been  precipitated  ihe  supernatant  salt 
solution  is  pipetted  off  and  discarded.  The  bacteria  are  mixed  with  the  serum 
of  an  animal  immunized  against  the  same  organism.  This  mixture  is  incubated 
at37°C.for  24  hours  and  then  centrifugalized  until  bacteria  precipitate.  The 
serum  is  syphoned  off  and  discarded.  The  bacteria  are  washed  in  several 
changes  of  normal  salt  solution  to  rid  them  of  serum.  They  are  mixed  with 
sufficient  salt  solution  to  make  a  suspension  containing  the  desired  amount  of 
bacteria  per  cubic  centimeter;  and  are  then  sterilized  in  a  water  bath  at  55°C. 
to  6o°C.  for  Y2  hour. 

This  sensitization,  when  successfully  carried  out,  is  said  to  prevent  or  mini- 
mize undesirable  reactions  following  the  injection  of  the  vaccine,  to  prevent  the 
occurrence  of  a  negative  phase  to  permit  the  administration  of  larger  doses  and 
repetition  of  doses  at  shorter  intervals  and  to  produce  a  higher  degree  of  im- 
munity than  it  is  possible  to  secure  with  non-sensitized  vaccines. 

TUBERCULIN 

Tuberculin  is  used  by  some  in  the  treatment  and  in  the  diagnosis  of 
tuberculosis. 

From  among  the  various  tuberculins,  the  selection  of  one  for  use  in  the  treat- 


214  MEDICAL  BACTERIOLOGY 

ment  of  a  particular  case  is  determined  by  the  signs,  symptoms,  localization, 
extent  and  progress  of  the  disease  and  by  the  fancies  of  the  person  using  the 
tuberculin. 

Tuberculin  is  of  greatest  value  as  a  diagnostic  agent. 

Koch's  Old  Tuberculin.— (Tuberculinum  original  alt)  "T.  O.  A."  or  "O. 
T."  is  made  by  planting  the  tubercle  bacillus  in  a  flask  of  glycerin  broth, 
incubating  at  37°C.  for  8  weeks.  It  is  then  placed  in  steam  sterilizer  for  i 
hour  or  autoclave  at  1 5  pounds  pressure  for  J^  hour  and  evaporated  on  a  water 
bath  at  80° C.  to  Ko  its  original  volume.  The  bacteria  are  removed  by  nitra- 
tion, first  through  paper  and  then  through  a  Berkefeld  filter,  the  filtrate,  which 
is  the  tuberculin,  is  put  in  containers  and  sterilized  in  steam  sterilizer,  i  hour 
each  day  for  3  successive  days,  or  in  the  autoclave  at  15  pounds  pressure  for 
20  minutes. 

Deny's  Bouillon  Filtrate.— (Bouillon  Filtrate)  "B.  F."  is  prepared  the  same 
as  O.  T.  except  that  it  is  not  heated  at  any  time  during  its  preparation.  It  is 
evaporated  to  one-tenth  its  original  volume  at  room  temperature  in  vacua  over 
sulphuric  acid. 

Koch's  Bacillen  Emulsion.— "B.  E."  cultures  are  made  as  for  the  prepara- 
tion of  O.  T.,  the  pellicle  of  bacteria  is  caught  on  cheesecloth  as  the  culture  is 
poured  on  it,  they  are  freed  of  broth  by  pouring  over  them  first  salt  solution, 
then  water.  The  bacteria  are  placed  in  dishes  and  desiccated  in  vacuo  over 
sulphuric  acid  and  weighed,  then  ground  in  a  porcelain  ball  mill  for  about  6 
weeks;  100  cc.  of  salt  solution  for  each  gram  of  bacteria  is  then  put  in  the  mill 
and  grinding  continued  for  2  days.  The  emulsion  is  allowed  to  stand  at  rest 
for  several  days  so  that  coarser  particles  precipitate,  the  supernatant  fluid  is 
then  collected,  mixed  with  50  per  cent,  of  glycerin  and  standardized  to  contain 
5  mg.  of  solids  in  each  cubic  centimeter. 

Tuberculin  Residue  "T.  R."  ("New  tuberculin"). — Bacilli  are  grown  and 
recovered  from  the  culture,  dried,  weighed  and  powdered,  the  same  as  when 
proceeding  to  make  B.  E.  When  sufficiently  comminuted,  100  cc.  of  sterile 
distilled  water  is  added  to  each  gram  of  dried  bacteria,  and  the  mass  again 
ground  for  24  hours.  The  emulsion  is  then  centrifugalized  until  the  solids 
precipitate.  The  supernatant  fluid  is  siphoned  off  and  discarded.  The  solid 
residue  is  collected,  desiccated  in  vacuo  over  sulphuric  acid,  mixed  with  water 
and  after  24  hours  centrifugalized  as  before.  The  supernatant  fluid,  second 
watery  extract,  is  collected  and  set  aside;  the  residue  again  desiccated  in  vacuo, 
after  which  it  is  extracted  with  water  for  the  third  time,  the  same  as  before  and 
the  watery  extract  set  aside. 

By  repeating  in  the  same  way  several  times  more  this  extraction,  and  retain- 
ing the  extracts,  eventually  there  is  no  residue;  the  entire  bacillary  substance 
having  been  put  in  solution. 

These  several  water  extracts  (solutions)  are  then  mixed. 

During  the  process,  the  amount  of  water  added  each  time  to  residue  must  be 
regulated  so  that,  when  combined,  there  will  be  a  total  volume  100  cc.  of  solu- 
tion for  each  gram  of  bacteria  with  which  the  production  was  started. 


BACTERIAL  VACCINES  215 

A  measured  portion  of  the  combined  extracts  is  dried  in  vacuo  and  the  solid 
residue  weighed  to  determine  the  amount  of  solids  per  cubic  centimeter. 

Finally  glycerin  and  water  (equal  parts)  are  mixed  with  the  combined  watery 
extracts  so  that  each  cubic  centimeter  of  the  finished  product  contains  2  milli- 
grams of  solids. 

Tuberculin  is  made  from  the  human  tubercle  bacillus  and  from  the  bovine 
tubercle  bacillus. 

In  the  preparation  of  all  these  tuberculins  it  is  a  customary  precaution  to 
add  0.5  per  cent,  phenol  just  before  placing  in  containers  for  storage  or  dispens- 
ing and  when  diluting  for  use  0.5  per  cent,  phenol  solution  is  the  diluent 
employed. 

B.  F.,  B.  E.  and  T.  R.,  tuberculins  not  subjected  to  sterilization  by  heat,  are 
tested  for  sterility  by  planting  on  glycerin  agar  slants  and  guinea-pig  injections 
before  dispensing. 

To  determine  whether  grinding  of  dried  bacteria  has  been  adequate,  sam- 
ples are  stained  and  examined  microscopically  and  grinding  continued  until 
such  examinations  reveal  no  bacteria  that  have  escaped  comminution. 

There  are  many  tuberculins  which  are  slight  modifications  of  those  intro- 
duced by  Koch,  such  as  Maragliano's,  basically  and  physiologically  the  same, 
possessing  slight  if  any  advantages  over  the  Koch  products  and  not  generally 
employed. 

Radically  different,  scientifically  of  interest  to  the  bacteriologist  and  im- 
munologist  and  perhaps  worthy  of  more  extensive  clinical  investigation  than 
has  been  given  it,  is  Von  Ruck's  tuberculin. 

In  the  Medical  Record,  vol.  Ixxxii,  Aug.  31,  1912,  Karl  Von  Ruck  describes  the  preparation, 
physiological  action  and  dosage  of  his  tuberculin  in  the  following  words : 

"Method  of  Preparing. — The  culture  of  tubercle  bacilli  used  is  of  human  origin,  grown  on 
bouillon,  and  has  been  perpetuated  in  my  laboratory  for  the  past  10  years;  it  was  apparently 
avirulent  when  first  tested  upon  guinea-pigs  and  has  continued  so  to  the  present  time.  On 
the  manufacture  of  the  preparation  heat  has  been  avoided  and  the  chemical  effect  of  light 
excluded.  No  chemicals  have  been  introduced  in  kind  or  concentration  that  could  injure, 
split,  reduce,  or  alter  the  several  constituents.  The  cultures  having  reached  their  maximum 
growth  are  collected  upon  a  filter  and  washed  free  of  adhering  culture  fluid  until  the  filtrate 
gives  no  further  biurette  reaction.  The  bacillary  mass  is  then  transferred  to  a  glass  container 
immersed  in  distilled  water  containing  0.4  per  cent,  phenol,  and  with  frequent  stirring  and 
shaking  it  is  macerated  for  several  days,  when  the  filtrate  obtained  contains  the  protein 
designated  as  No.  i;  chemically  examined,  it  shows  primary  proteose,  25  per  cent.;  secondary 
proteose  70  per  cent.;  peptone,  small  amount;  reaction  acid.  After  further  washing  with  dis- 
tilled water,  the  bacilli  are  dried  and  powdered,  when  their  fat  extraction  follows;  after  drying 
they  are  again  powdered  and  then  partially  extracted  in  distilled  water  yielding  protein  No.  2, 
this  showing  coagulable  protein,  0.03  per  cent,  (estimated  by  nitrogen) ;  primary  proteose  and 
deuterproteose  in  about  equal  amounts,  total  about  48  per  cent.,  secondary  proteose  50  per 
cent.,  peptone  trace,  phosphorous  content  o.  i  per  cent,  and  alkaline  reaction.  The  bacillary 
mass  is  again  dried  and  powdered  and  suspended  in  0.4  per  cent,  carbolized  distilled  water  and 
then  ground  wet  in  glass  capsules  with  agate  marble,  until  repeated  microscopic  examinations 
no  longer  show  a  fragment  or  formed  substance  of  the  bacilli.  Filtration  through  porcelain 
gives  protein  No.  3,  in  solution,  differing  from  No.  2  by  absence  of  coagulable  protein,  the 
relatively  small  amount  of  primary  and  increased  amount  of  secondary  proteose  (75  per  cent.) 


2l6  MEDICAL  BACTERIOLOGY 

peptone  is  shown  in  small  amount.  The  phosphorous  content  is  0.5  per  cent.,  the  reaction 
acid. 

"  Protein  No.  4  passes  the  filter  after  an  addition  of  an  0.4  per  cent,  solution  of  sodic  hydrate 
and  gives  all  reactions  for  nucleo  protein.  The  remaining  bacillary  residue  amounting  to  about 
5  per  cent,  weight  of  fat  from  tubercle  bacilli,  is  free  from  proteins  (nitrogen  determination), 
may  contain  traces  of  fats,  and  is  probably  cellulose.  The  secondary  proteoses  give  reaction 
for  sugar. 

"The  fatty  extractives  of  tubercle  bacilli  may  be  obtained  separately  as  neutral  fats  and 
fatty  acids  by  their  saponification  and  subsequent  extraction  of  the  acid  precipitate  with  alco- 
hol and  with  ether,  or  in  order  to  avoid  undesirable  chemical  changes  by  saponification,  the 
alcohol  fats  and  the  ether  fats  may  be  obtained  respectively  by  first  complete  extraction  with 
ether,  followed  by  alcohol  and  vice  versa,  which  to  me  seems  preferable.  When  in  solution  they 
can  be  shaken  out  with  distilled  water,  the  later  holding  about  0.5  per  cent,  in  free  suspension, 
which  is  opalescent  in  appearance  in  that  concentration,  the  opalescence  disappears  in  the  con- 
centration employed  by  me.  The  several  proteins  and  fatty  extractives  having  been  obtained 
separately  and  having  each  been  standardized,  any  free  alkali  in  protein  No.  4  having  been 
neutralized  by  addition  of  weak  solution  of  HC1,  just  short  of  causing  precipitation,  the  several 
constituents  are  assembled  to  represent  the  formula  (given  below). 

"The  precautions  employed  in  preventing  injury  or  undesirable  modifications  of  the  several 
bacillary  products  have  not  all  been  demonstrated  as  absolutely  requisite  for  their  highest 
efficiency,  my  purpose  in  their  adoption  having  been  to  take  no  chances  of  an  inferior  prepara- 
tion by  their  omission. 

"Formula. — This  vaccine  is  absolutely  free  from  real  or  supposed  danger,  powerful  enough 
to  act  by  one  or  two  applications,  uniform  in  action  by  producing  the  desired  immunity  in  all 
cases,  simple  enough  to  make  it  available  for  use  of  the  general  practitioner,  without  the 
necessity  of  elaborate  examinations  or  investigations  in  selecting  suitable  cases. 

"The  vaccine  ready  for  use  contains  per  cubic  centimeter  10  milligrams  of  proteins  of 
tubercle  bacilli  and  a  small  amount  of  their  fatty  extractives,  proportioned  quantitatively 
as  follows: 

Protein  No.  i,  o.  25  mg. 
Protein  No.  2,  2.75  mg. 
Protein  No.  3,  i .  oo  mg. 
Protein  No.  4,  6 .  oo  mg. 
Fatty  extractives,  o.oi  mg. 

In  the  adoption  of  the  amounts  of  the  several  constituents  present  in  the  preparation,  I  was 
governed  by  the  study  of  their  action  when  employed  separately  in  animal  experiments  as  well 
as  in  some  children.  Finding  that  with  the  same  doses  but  little  effect  was  noted  in  the  produc- 
tion of  specific  antibodies  in  the  use  of  protein  No.  i,  this  is  present  in  the  smallest  amount, 
whereas  protein  No.  2,  and  especially  the  nucleoprotein  designated  as  No.  4,  appearing  most 
effective,  they  are  quantitatively  in  excess.  The  addition  of  fatty  extractives  may  be  even- 
tually found  unnecessary,  it  appearing  in  my  studies  of  complement  fixation  with  the  several 
constituents  of  the  tubercle  bacillus  as  antigens,  that  such  fixation  occurs  with  all  of  them 
inclusive  of  the  fatty  extractives  with  the  sera  of  non-tuberculous  children  after  their  vaccina- 
tion with  protein  Nos.  2,  3  and  4,  irrespective  of  the  presence  of  one  or  the  other  of  the  fatty 
extractives  in  the  preparation. 
"The  dose  of  vaccine  is: 

0.05  to  o.  10  to  nurslings. 

0.02  to  0.60  cc.  to  children.     The  higher  dosage  is  given  to  those  18  to  20  years  old. 

"Usually  at  point  of  injection  in  arm  there  isn't  much  swelling  or  tenderness  unless  the 
patient  is  suffering  from  an  active  tuberculous  lesion." 


BACTERIAL  VACCINES  217 

In  properly  selected  cases,  when  properly  administered,  tuberculin  will  often 
establish  the  nature  of  a  tubercular  infection  when  other  methods  of  diagnosis 
fail  to. 

Tuberculin,  like  other  bacterial  products,  is  a  potent  agent  capable  of  bene- 
fiting or  seriously  injuring  those  treated  with  it. 

It  should  only  be  administered  by  those  familiar  with  its  properties  and  ex- 
perienced in  immunology. 

Koch's  old  tuberculin  is  the  tuberculin  of  choice  for  diagnostic  purposes. 
There  are  different  methods  of  using  it.  Its  value  is  based  on  the  fact  that, 
when  properly  administered,  a  localized  or  general  reaction  occurs  in  tuberculous 
patients  and  no  reaction  occurs  in  the  non- tuberculous. 

For  diagnostic  purposes  tuberculin  is  injected  subcutaneously,  rubbed  into 
a  scarified  cutaneous  surface,  rubbed  into  the  skin  where  there  is  no  breach  of 
continuity,  or  dropped  into  the  eye.  Special  advantages  are  claimed  for  each 
of  these  methods  and  each  has  its  limitations. 

The  subcutaneous  administration  of  tuberculin  gives  the  most  accurate  re- 
sults. It  is  strictly  contraindicated  when  the  patient's  temperature  is  above 
normal  and  perhaps  should  not  be  used  to  diagnosticate  tuberculosis  in  children 
under  10  years  of  age.  Successful  employment  depends  upon  accurate  observa- 
tion of  the  patient's  temperature  and  physical  signs  before  and  after  the  injec- 
tion of  tuberculin. 

The  degree  of  reaction  manifest  by  a  tuberculous  patient  following  an  injec- 
tion depends  upon  the  sensitiveness  of  the  patient  and  the  size  of  the  dose. 
This  reaction  may  be  nothing  more  than  inflammation  at  the  point  of  inocula- 
tion, some  or  all  of  the  symptoms  and  physical  signs  of  disease  may  be  aggra- 
vated, there  may  be  a  rise  of  temperature  from  o.5°F.  to  2°F.,  or,  in  a  severe  reac- 
tion, all  these  manifestations  may  be  observed. 

The  endeavor  of  the  diagnostician  is  to  obtain  a  distinct  reaction,  but  as  slight 
a  one  as  possible;  a  rise  of  temperature  greater  than  i°F.  is  always  to  be  avoided. 

Different  patients  vary  to  a  great  degree  in  sensitiveness  to  tuberculin,  hence 
the  initial  dose  must  be  less  than  could  injure  the  most  sensitive,  consequently 
the  first,  perhaps  the  second  injection,  will  not  cause  a  reaction  in  the  majority 
of  tuberculous  patients. 

The  technique  of  the  subcutaneous  tuberculin  test  is  as  follows: 

The  patient's  temperature  is  taken  every  2  hours  for  i  or  2  days  before  in- 
jecting and  symptoms  and  physical  signs  are  carefully  noted. 

The  first  injection  should  be  o.oooi  cc.  or  less.  Following  the  injection  the 
patient's  temperature  should  be  taken  and  the  physical  signs  noted  every  2  or 
3  hours  for  24  hours.  If  no  reaction  is  caused  by  the  first  injection  a  second 
test  is  made  after  an  interval  of  48  hours,  using  o.ooi  cc.  If  the  second  test  is 
negative,  subsequent  tests  are  made  at  intervals  of  48  hours,  using  0.002  cc., 
0.003,  °-o°4>  o-°°5>  0.007,  0.009. 

Should  there  be  reason  to  suspect  tuberculosis,  in  spite  of  negative  reactions 
obtained  with  tuberculin  from  the  human  type,  then  the  tests  may  be  repeated, 
using  tuberculin  from  the  bovine  type  of  the  tubercle  bacillus. 


2l8  MEDICAL  BACTERIOLOGY 

Von  Pirquet  Test. — For  this  test  a  mixture  of  Koch's  tuberculin  i  part,  5 
per  cent,  aqueous  solution  phenol  i  part,  and  normal  salt  solution  i  part  is  used. 

The  patient's  arm  is  cleansed  with  ether  and  three  areas  about  %  inch  in 
diameter  are  scarified.  Two  drops  of  tuberculin  are  placed  upon  two  of  these 
areas  and  allowed  to  dry  in,  care  is  taken  that  no  tuberculin  touches  the  third, 
the  control  area. 

The  scarified  areas  must  be  at  least  2  inches  apart  and  the  degree  of  scarifica- 
tion must  be  exactly  the  same  in  each  instance.  Only  the  epiderm  is  scarified, 
blood  must  not  be  drawn.  A  positive  reaction  shows  redness  and  edema  of  the 
areas  inoculated  with  tuberculin  48  hours  after  introduction,  a  papule  or  vesicle 
follows  and  subsides  in  about  a  week.  The  control  area  shows  none  of  these 
changes. 

The  results  of  the  work  of  the  last  couple  of  years  suggest  that  in  the  near 
future  the  intracutaneous  tuberculin  test  as  carried  out  by  Craig  of  London, 
Canada,  will  supplant  the  Von  Pirquet.  This  is  substantially  as  follows: 

The  patient's  arm  is  cleansed  as  for  a  Von  Pirquet.  A  drop  of  sterile  glycerin 
broth  is  placed  on  the  skin  and  driven  in  by  puncturing  the  epidermis  six  times 
with  a  round,  solid,  sewing  needle.  Several  inches  away  a  drop  of  tuberculin 
is  placed  on  the  skin  and  punctures  are  made  through  this.  Excess  broth  and 
excess  tuberculin  are  wiped  away.  In  making  the  punctures,  care  is  taken  to 
thrust  the  needle  point  into  but  not  through  the  skin,  and  blood  should  not  be 
drawn. 

Moro  Test. — A  50  per  cent,  lanolin  ointment  of  old  tuberculin  is  rubbed  into 
the  skin  of  the  abdomen  over  an  area  of  about  6  square  centimeters.  A  positive 
reaction  shows  a  red  papular  eruption  after  i  or  2  days. 

Both  the  Von  Pirquet  and  Moro  test  have  their  greatest  value  when  applied 
to  young  children.  A  large  number  of  apparently  healthy  adults  show  positive 
reactions  when  subjected  to  these  tests. 

Calmette's  Ophthalmo  Test. — Calmette's  ophthalmo  test  is  performed  by 
dropping  into  one  eye  i  minim  of  a  i  per  cent,  aqueous  solution  of  purified  tuber- 
culin^ twice  precipitated  with  alcohol). 

This  test  frequently  produces  misleading  results  and  is  not  without  danger. 

RABIES  (Lyssa,  Hydrophobia) 

Rabies  is  a  disease,  primarily  of  dogs  and  wolves,  which  occurs  in  epizootic 
form,  in  cats,  cows,  horses,  swine,  sheep,  deer  and  various  wild  animals. 

In  man  the  disease  occurs  as  a  result  of  infection  by  the  bite  of  a  rabid  ani- 
mal— in  the  vast  majority  of  cases  dogs  or  wolves  being  the  offenders. 

The  bite  of  animals  afflicted  with  this  disease  is  infectious  from  i  week  before 
they  present  signs  of  illness  until  the  termination  of  the  disease. 

Not  all  animals  suspected  have  the  disease,  and  fortunately,  only  a  portion 
of  those  bitten  by  rabid  animals  are  infected. 

Rabies  is  hopeless  in  man,  once  clinical  signs  appear,  but  it  is  possible  to 
immunize  persons  infected,  before  the  termination  of  the  period  of  incubation, 


BACTERIAL  VACCINES  2IQ 


provided  prophylactic  vaccination  is  instituted  shortly  after  infection.  One 
can  only  determine  whether  or  not  an  animal  that  has  bitten  a  person  has  the 
disease,  by  microscopic  examination  of  the  brain,  and  subdural  inoculation  of 
rabbits  with  several  drops  of  brain  emulsion. 

When  a  suspected  animal  is  killed  for  such  an  examination,  injury  of  its 
head  should  be  avoided.  Decapitation  should  be  done  so  as  to  leave  as  much 
as  possible  of  the  neck  attached  to  the  head.  If  the  head  must  be  held  for  some 
hours  or  shipped  before  examination,  it  should  be  packed  in  a  bucket  of  ice. . 

The  entire  calvarium  and  posterior  portion  of  the  cervical  vertebrae  are  sawed 
away  and  removed  without  injury  of  underlying  tissue  before  any  attempt  is 
made  to  remove  the  bram  and  medulla. 

Films  for  Microscopic  Examination  are  prepared  as  follows :  An  incision  is 
made  at  right  angles  to  the  surface  and  to  the  long  axis  of  convolution  into  the 
hippocampus  major  and  cerebellum.  Pieces  of  tissue  about  i  millimeter  thick 
and  several  millimeters  in  diameter  are  removed,  each  placed  near  the  end  of  a 
glass  slide  and  spread  in  a  thin  even  film  by  sliding  a  second  slide  over  the  first 
one  while  making  gentle  pressure.  They  are  immediately  immersed  in  methyl 
alcohol  for  fixation.  This  must  be  done  rapidly  to  prevent  drying  and  conse- 
quent tissue  changes.  After  3  minutes  in  alcohol  slides  are  removed  and  stained 
for  30  seconds  with  the  following  stain,  freshly  prepared. 

Engle's  modification  of  Van  Gieson's  stain  for  negri  bodies: 

LoefHer's  alkaline  methylene  blue 5  cc. 

Distilled  water 20  cc. 

Saturated  alcoholic  sol.  fuchsin 4  drops. 

When  properly  stained  the  protoplasm  of  nerve  cells  is  faint  blue,  nucleus  purple 
and  nucleolus  dark  blue.  If  the  blue  is  too  intense  the  staining  may  be  corrected 
by  adding  more  distilled  water  to  the  stain  and  heating  slides  while  staining. 
Negri  bodies  occur  within  the  protoplasm  of  nerve  cells,  usually  one,  occasion- 
ally several,  appear  in  one  nerve  cell.  They  stain  maroon  red  and  contain  one 
or  more  deep  purple  or  black  inner  bodies. 

Negri  bodies,  first  described  by  A.  Negri  in  1903,  are  found  in  almost  99  per- 
cent, of  smears  from  brains  of  rabid  animals,  they  have  never  been  found  in 
other  diseases;  their  nature  is  obscure. 

Negri  bodies  are  round  or  oval  and  vary  in  size  from  i  fj,  to  20  jj,  in  diameter. 
The  smallest  are  observed  in  brain  cells  of  rabbits  inoculated  with  virus  fixe; 
the  largest  in  the  brain  cells  of  cows  afflicted  with  rabies.  In  the  brains  of  dogs 
dead  from  street  infection  they  vary  from  barely  visible  to  about  10  p. 

Inoculation  of  Rabbits. — As  soon  as  specimens  have  been  removed  from  a 
suspected  brain  for  microscopic  examination,  the  brain  should  be  placed  in 
.glycerin.  If  microscopic  examination  fails  to  disclose  negri  bodies,  remove  brain 
from  glycerin,  excise  from  beneath  the  surface  a  piece  of  cerebellum  about  one- 
half  the  size  of  a  walnut,  place  in  a  sterile  glass  mortar  with  15  or  20  cc.  of  sterile 
normal  salt  solution  and  emulsify  by  trituration.  Allow  the  suspension  to  stand 


22O  MEDICAL  BACTERIOLOGY 

for  a  few  minutes  so  that  gross  particles  precipitate,  then  draw  several  cubic 
centimeters  into  a  glass  syringe. 

Have  an  assistant  hold  a  rabbit.  With  a  sharp  scalpel  make  an  incision 
about  ^  inch  long  through  the  skin  in  the  median  line  midway  between  the  eyes. 
Bore  a  small  hole  through  the  bone  and  thrust  the  tip  of  the  needle  through  the 
dura  and  inject  several  drops  with  the  syringe.  Seal  the  incision  with  collodion 
and  place  rabbit  in  a  closed  observation  cage.  The  period  of  incubation  is 
usually  less  than  3  weeks,  occasionally  it  is  prolonged. 

The  onset  of  the  disease  may  be  marked  by  maniacal  excitement  followed  in 
24  to  72  hours  by  progressive  paralysis  and  death.  The  stage  of  excitement  may 
not  occur,  the  first  evidence  of  the  disease  being  paratysis  of  the  hind  quarters 
and  muscles  of  deglutition — "dumb  rabies" — the  paralysis  progressing  and 
terminating  fatally  in  3  to  5  days. 

The  specific  cause  of  rabies  is  still  a  mooted  point.  Some  believe  the  negri 
bodies  the  specific  cause  (not  likely).  F.  Proescher  and  others  have  isolated 
from  the  brain  and  cord  of  rabid  animals  a  small  coccus  which  may  or  may  not 
be  the  cause.  Probably  not. 

The  causative  virus,  whatever  its  nature,  is  present  in  the  saliva  and  in  the 
spinal  cord  and  brain  of  infected  animals  several  days  before  the  onset  of  the 
disease.  It  resists  antiformin  and  glycerin,  and  can  be  transmitted  from  animal 
to  animal. 

Pasteur  discovered  that  after  passing  the  virus  from  one  rabbit  to  another 
through  a  series  of  about  40  or  50  rabbits,  the  virus  finally  acquires  certain  new, 
permanent  properties  and  is  then  known  as  "  Virus  Fixe." 

Rabies  vaccine  is  made  from  the  spinal  cord  or  brain,  removed  from  a  rabbit 
dead  from  the  effect  of  inoculation  with  virus  fixe. 

The  amount  of  virus  in  such  brain  or  cord  is  attenuated  by  suspending  it  in 
a  glass  jar  over  a  layer  of  potassium  hydrate  at  22°C.  The  longer  it  is  so  dried 
the  greater  the  attenuation,  until  the  virus  entirely  disappears,  which  it  will  do 
in  from  3  to  4  weeks,  according  to  the  thickness  of  the  cord. 

Pasteur's  method  of  administering  rabies  vaccine  to  man  is  about  as  follows: 

Day  of  treatment  Cord  that  has  been  dried,  Amount 

days 

ist  day,  10  A  M.  14  or  13  3 

ist  day,    4  P.M.  12  or  n  3 

2d  day,  10  A.M.  10  or  6                              3 

2d   day,    4  P.M.  8  or  7                              3 

3d   day,  10  A.M.  6  2 

3d   day,    4  P.M.  6  2 

4th  day,  10  A.M.  5  2 

5th  day,  10  A.M.  5  2 

6th  day,  10  A.M.  4  2 

yth  day,  10  A.M.  3  i 

8th  day,  10  A.M.  4  2 

Qth  day,  10  A.M.  3  i 

loth  day,  10  A.M.  5  2 

nth  day,  10  A.M.  5  2 

1 2th  day,  10  A.M.  4  2 


BACTERIAL  VACCINES  221 

Day  of  treatment  Cord  that  has  been  dried.  Amount 

days 

1 3th  day,  10  A.M.  4  2 

1 4th  day,  10  A.M.  3  2 

1 5th  day  5  2 

1 6th  day  5  2 

1 7th  day  4  2 

1 8th  day  4  2 

igth  day  3  2 

2oth  day  3  2 

2ist  day  3  i 

When  the  above  method  of  treatment  is  to  be  carried  out,  a  cord  which  has 
been  dried  for  the  designated  number  of  days  is  removed  from  the  jar,  about  a 
third  of  it  cut  off  and  ground  up  in  a  mortar  with  salt  solution  until  emulsified; 
the  emulsion  is  allowed  to  stand  at  rest  for  a  number  of  minutes  until  gross 
particles  precipitate,  then  the  amount  of  the  supernatant  fluid  to  be  injected 
is  drawn  into  a  hypodermic  syringe  and  injected  into  the  abdominal  wall.  In 
recent  years  it  has  been  shown  that  an  emulsion  of  undried  brain  or  cord,  con- 
taining many  times  the  total  amount  of  virus  injected  in  3  weeks  by  the  Pasteur 
method,  may  be  given  as  a  first  injection  without  producing  any  untoward 
effects,  and  without  danger  of  producing  rabies.  At  the  present  time,  in  some 
hospitals,  treatment  is  completed  in  5  days  by  giving  daily  injections  of  an 
emulsion  of  undried  brain  or  cord. 

Rabies  vaccine  is  employed  to  immunize  against  rabies.  The  treatment 
should  be  instituted  at  the  earliest  possible  moment  after  a  probable  infection. 

If  rabies  vaccine  is  not  given  in  time  to  produce  immunity  before  the  onset 
of  symptoms  of  the  disease  it  is  useless. 

SMALL-POX  VACCINE 

Small-pox  vaccine  is  the  serum  obtained  from  a  vesicle  on  the  skin  of  a  calf 
suffering  with  cow  pox. 

This  serum  is  employed  to  produce  immunity  against  small  pox.  The  arm 
of  the  person  to  be  inoculated  is  washed  with  soap  and  water  and  alcohol, 
dried  and  scarified  without  drawing  blood.  Several  drops  of  the  serum  are 
rubbed  into  the  scarified  area  and  allowed  to  dry  there. 

When  the  desired  effect  occurs,  redness  and  swelling  develops  at  the  point  of 
inoculation  in  about  3  days;  several  days  later  a  vesicle  appears;  this  is  trans- 
formed into  a  pustule  in  the  course  of  several  days,  and  the  pustule  gradually 
dries,  a  dark  scab  forms  and  healing  occurs  so  that  the  entire  process  disappears 
in  from  8  to  16  days. 

COLEY'S  FLUID 

Coley's  fluid  is  used  in  the  treatment  of  inoperable  cases  of  sarcoma.  It  is 
injected  subcutaneously  at  the  margin  of  the  tumor  or  at  any  other  point.  The 
initial  dose  is  Y±  minim;  in  the  course  of  several  hours  a  marked  reaction  of 
short  duration  follows. 


222  MEDICAL  BACTERIOLOGY 

An  injection  is  given  daily,  the  dose  being  increased  as  rapidly  as  possible 
without  producing  reactions. 

Grow  bacillus  prodigiosus  on  agar  at  room  temperature  for  10  days.  Scrape 
off  the  growth  with  a  glass  rod  and  rub  to  a  paste  in  a  glass  mortar,  using  normal 
salt  solution  as  diluent,  bottle  and  sterilize  at  75°C.  for  i  hour.  Place  in  ice 
box.  Grow  virulent  streptococci  in  bouillon  at  37°C.  for  10  days. 

Determine  the  amount  of  nitrogen  per  cubic  centimeter  in  the  prodigiosus 
paste  to  ascertain  the  amount  of  protein  per  cubic  centimeter  and  so  dilute  it 
with  normal  salt  solution  that  each  cubic  centimeter  of  the  vaccine  will  contain 
2.5  mg.  of  protein. 

To  each  100  cc.  of  the  bouillon  culture  of  streptococci  add  30  cc.  of  prodigio- 
sus suspension  and  20  cc.  of  glycerin.  Place  in  bottles,  add  a  crystal  of  thymol 
to  each  and  sterilize  in  a  water  bath  at  75°C.  for  2  hours. 

HAFFKINE'S  VACCINE 

Haffkine's  vaccine  is  made  by  planting  the  bacillus  pestis  in  plain  bouillon 
to  which  sufficient  sterile  oil  or  butter  has  been  added  to  cover  the  surface  with 
numerous  droplets.  The  culture  is  incubated  at  35°C.  to  37°C.  where  there  is 
little  or  no  vibration.  Growth  occurs  around  and  adheres  to  the  droplets  of 
oil,  forming  white  stalactites  that  hang  from  the  surface  as  icicles  hang  from  the 
roof  of  a  tunnel.  When  a  crop  of  stalactites  forms,  the  flask  is  shaken  and  they 
fall  to  the  bottom. 

The  flask  is  again  incubated  and  a  second  crop  of  stalactites  forms  and  is 
shaken  down. 

When  the  fifth  or  sixth  crop  of  stalactites  has  formed  the  flask  is  shaken  until 
they  are  broken  up  and  the  bacteria  evenly  distributed  throughout  the  medium. 
The  flask  is  then  submerged  in  a  water  bath  and  heated  at  75°C.  for  2  hours. 
The  vaccine  is  then  ready  for  use. 

Haffkine's  vaccine  is  administered  to  healthy  people  to  immunize  them 
against  plague.  It  confers  almost  complete  immunity  to  the  bubonic  but  only 
slight  immunity  to  the  pneumonic  form  of  plague. 

Two  subcutaneous  injections  are  given,  the  first  2  cc.  to  3  cc.  and  10  to  15 
days  later  from  3  cc.  to  5  cc. 

ANTHRAX  VACCINE 

Plain  broth  cultures  are  incubated  at  42°C.  until  they  become  avirulent  for 
rabbits  and  guinea-pigs  but  are  still  lethal  for  mice.  From  cultures  so  at- 
tenuated, vaccine  No.  i  is  made,  it  being  a  subculture  in  broth  inoculated  at 
37-5°C. 

Other  cultures  are  attenuated  by  incubation  at  42°C.  until  avirulent  for 
rabbits  but  still  lethal  for  both  guinea-pigs  and  mice.  From  cultures  of  this 
attenuation  vaccine  No.  2  is  made,  it  being  48-hour-old  subculture  in  broth 
incubated  at  37-5°C. 

Cattle  and  sheep  are  successfully  immunized  to  infection  with  the  anthrax 
bacillus  by  two  subcutaneous  injections  a  fortnight  apart.  Vaccine  No.  i  is 
employed  for  the  first  injection  and  vaccine  No.  2  for  the  second.  It  is  not 
employed  in  homo. 


CHAPTER  VII 

THERAPEUTIC  SERA 

Diphtheria  Antitoxin 

When  a  meat-infusion  bouillon  culture  of  the  diphtheria  bacillus,  which  has 
been  incubated  at  37°C.  for  several  weeks,  is  filtered  through  a  porcelain  filter, 
the  filtrate  contains  the  toxin,  but  no  bacteria,  and  we  speak  of  this  filtrate 
as  toxin.  It  is  capable  of  producing  disease  and  death  when  injected  into 
susceptible  animals  in  sufficient  quantity. 

Different  cultures  of  diphtheria  bacillus  produce  different  quantities  of  toxin, 
consequently  when  toxin  is  obtained  by  filtering  a  bouillon  culture,  its  strength 
must  be  determined.  A  unit  of  diphtheria  toxin  is  the  smallest  amount  that 
will  regularly  kill  a  25o-Gm.  guinea-pig  in  4  days;  this  amount  is  called  the 
"Minimum  Lethal  Dose,"  expressed  "M.L.D." 

The  first  step  in  the  production  of  diphtheria  antitoxin  is  to  produce  toxin 
and  determine  its  M.L.D.  The  next  is  to  immunize  a  horse.  This  is  done  by 
injecting  a  minute  quantity  of  the  toxin  into  a  horse.  About  a  week  after  the 
first  injection  the  horse  is  less  susceptible  to  the  toxin  than  before  and  a  second 
injection,  larger  than  the  first  in  amount,  is  given.  At  intervals  of  about  a 
week  subsequent  injections  of  gradually  increasing  amounts  of  toxin  are  given 
until,  in  2  or  3  months,  the  horse  acquires  maximum  immunity.  Then,  under 
aseptic  conditions,  the  horse  is  bled  and  the  serum  obtained  from  the  blood. 
The  antitoxic  properties  of  the  serum  are  confined  to  the  serum  pseudoglobulins, 
the  pseudoglobulins  are  removed  from  the  serum  by  mixing  the  serum  with  an 
equal  amount  of  saturated  ammonium  sulphate  solution;  this  is  allowed  to 
stand  for  12  hours  and  the  precipitate  is  then  collected  on  hard  filter-paper. 

The  precipitate  is  dissolved  in  sufficient  water  to  bring  it  up  to  the  original 
volume  of  the  serum,  an  equal  amount  of  saturated  ammonium  sulphate 
solution  is  added  and,  after  12  hours,  the  precipitate  is  collected  and  dried  be- 
tween filter-papers  until  it  has  a  pasty  consistency.  It  is  then  dissolved  in  a 
saturated  solution  of  sodium  chloride.  This  solution  is  filtered  and  2.5  cc.  of 
80  per  cent,  acetic  acid  is  added  to  each  liter  of  the  filtrate.  The  precipitate  is 
collected  and  dried  on  filter-papers  and  dialyzed  in  running  water  until  dissolved. 
It  is  then  neutralized  and  again  dialyzed  until  free  of  sodium  chloride. 

The  amount  of  antitoxin  present  in  the  serum  of  immunized  horses  varies, 
hence  when  serum  is  obtained  its  strength  must  be  determined.  This  is  done 
by  mixing  various  amounts  of  serum  with  a  M.L.D.  of  toxin  and  injecting  the 
mixtures  into  guinea-pigs,  until  an  amount  is  found  which  is  just  sufficient  to 
neutralize  a  M.L.D.  of  toxin. 

223 


224  MEDICAL  BACTERIOLOGY 

A  "unit"  of  diphtheria  antitoxin  is  200  times  the  amount  necessary  to  neu- 
tralize one  M.L.D.  of  toxin. 

Diphtheria  antitoxin  is  employed  as  a  prophylactic  and  as  a  curative  agent. 

When  given  as  a  prophylactic  the  immunity  to  infection  conferred  is  a 
passive  immunity  and  hence  lasts  only  a  few  weeks. 

Diphtheria  antitoxin  is  specific  for  the  treatment  of  diphtheria.  The  earlier 
it  is  given  in  the  disease  the  better  the  results. 

When  given  as  a  prophylactic  and  in  mild,  early  cases  of  the  disease  it  is 
injected  subcutaneously,  preferably  in  the  left  side  of  the  abdominal  wall. 
In  late,  severe  cases  it  is  injected  into  a  vein. 

The  prophylactic  dose  is  from  1000  to  2000  units,  the  curative  dose  from 
10,000  to  40,000  units;  repeated,  if  necessary. 

TETANUS  ANTITOXIN 

Tetanus  antitoxin  is  produced  in  practically  the  same  way  as  diphtheria 
antitoxin.  Selected  horses  are  injected  at  intervals  with  increasing  amounts  of 
tetanus  toxin  (nitrate  from  bouillon  culture  of  tetanus  bacillus)  until  maximum 
immunity  is  acquired.  The  horse  is  then  bled  and  the  blood  serum  collected. 

The  American  unit  of  tetanus  antitoxin  is  10  times  the  amount  necessary  to 
immunize  a  35o-Gm.  guinea-pig  against  100  times  the  M.L.D.  of  toxin. 

Tetanus  antitoxin  has  its  greatest  value  when  employed  as  a  prophylactic, 
injected  subcutaneously  near  the  wound  as  soon  as  possible  after  the  occurrence 
of  a  wound  or  trauma  likely  to  be  infected  with  the  tetanus  bacillus,  such  as 
black  powder  burns,  deep  puncture  wounds  inflicted  by  splinters  of  wood  from 
old  buildings,  rusty  nails  or  instruments  contaminated  with  garden  soil,  stable 
dirt  or  hide  dust. 

The  prophylactic  dose  is  from  1000  to  2000  units;  this  may  be  repeated  in  a 
week  if  deemed  necessary. 

When  symptoms  of  tetanus  have  developed,  antitoxin  seldom  prevents  a 
fatal  result;  it  must  be  used  in  heroic  doses,  preferably  injected  into  the  spinal 
canal. 

ANTISTREPTOCOCCUS  SERUM 

Antistreptococcus  serum  is  produced  by  injecting  horses  with  killed  and 
attenuated  streptococci  until  maximum  immunity  is  acquired.  As  many  differ- 
ent strains  of  streptococci  as  can  be  procured  are  used,  to  produce  a  polyvalent 
serum. 

The  immunizing  power  of  such  serum  is  said  to  depend  upon  the  opsonin 
content. 

No  definite  dosage  has  been  established.  Large  quantities  are  required  to 
obtain  therapeutic  effect,  an  initial  dose  of  100  to  200  cc.,  followed  by  repeated 
doses  of  10  to  20  cc.  every  4  hours,  if  the  first  dose  proves  beneficial. 

Antistreptococcus  serum  should  be  injected  deep  into  the  muscles. 

The  indications  for  its  employment  are  said  to  be  acute,  severe  primary  and 
secondary  streptococcus  infection. 


THERAPEUTIC    SERA  225 

Antistreptococcus  serum  does  not  have  the  value  of  some  other  antitoxic 
and  bacteriocidal  sera.  Sometimes  it  produces  excellent  results  and  at  other 
times  it  exerts  no  influence  on  the  course  of  a  streptococcus  infection.  ' 

ANTIMENINGOCOCCUS  SERUM 

Antimeningococcus  serum  is  produced  by  injecting  horses,  alternately,  with 
killed  meningococci  and  solutions  prepared  as  follows: 

Pour  10  to  20  cc.  of  normal  salt  solution  on  a  24-hour-old  culture  of  meningo- 
cocci, shake  until  the  bacteria  are  suspended  in  the  salt  solution,  place  this  salt 
solution  suspension  in  the  incubator  at  37°C.  for  i  day  and  then  inject.  After 
the  horse  has  been  injected  at  intervals  for  a  couple  of  months  with  killed 
meningococci,  he  is  then  injected  with  living  meningococci  for  about  6  months. 

Antimeningococcus  serum  is  the  specific  for  the  treatment  of  spinal  men- 
ingitis caused  by  the  meningococcus.  It  is  injected  into  the  spinal  canal,  the 
quantity  being  determined  and  only  limited  by  the  patient's  tolerance,  from  10 
to  50  cc. 

ANTIANTHRAX  SERUM 
(SCLAVO'S  SERUM) 

Sclavo's  serum  is  produced  by  injecting  sheep  or  horses  first  with  sensitized 
bacilli  of  attenuated  virulence,  then  with  attenuated  bacilli,  not  sensitized, 
finally  with  virulent  bacilli. 

The  serum  of  animals  so  immunized  is  of  great  value  in  the  treatment  of 
anthrax.  It  should  be  employed  as  early  as  possible.  The  amount  used  is 
large,  from  20  to  100  cc.  repeated  if  necessary.  A  marked  reaction  follows  the 
first  injection  of  Sclavo's  serum.  In  grave  cases  and  those  coming  under  treat- 
ment late  in  the  disease,  the  serum  may  be  injected  into  a  vein;  in  less  urgent 
cases  intramuscular  or  subcutaneous  administration  is  practiced. 

When  injected  subcutaneously  it  takes  about  10  months  to  immunize  a 
horse  so  that  it  may  be  bled.  Amos  and  Wollstein  report  a  quicker  method. 
By  giving  intravenous  injections  of  the  autolized  products  of  meningococci  and 
para-meningococci,  and  living  virulent  cultures  of  these  organisms,  they  have 
produced  therapeutic  serum  in  3  months. 


CHAPTER  VIII 

WASSERMANN  AND  OTHER  COMPLEMENT  FIXATION  TESTS 

APPARATUS 

The  apparatus  needed  for  complement  fixation  tests : 

An  electric  centrifuge,  a  hot-air  sterilizer,  a  steam  sterilizer  or  autoclave,  a 
balance  sensitive  to  o.i  Gm.,  an  incubator,  a  small  water  bath,  one  dozen  gradu- 
ated centrifuge  tubes  (15  cc.  capacity),  half  a  dozen  wide-mouthed  ground-glass- 
stoppered  bottles  of  500  cc.  capacity,  a  like  number  of  similar  bottles  of  200  cc. 
capacity,  50  florence  flasks  (100  cc.  capacity),  50  deep  petri  dishes  (4  inches  in 
diameter),  100  glass  vials  (2  cc.  capacity),  and  rubber  stoppers  for  same,  100 
test-tubes  of  each  of  the  following  sizes:  6  by  i  inch,  6  by  %  inch,  4  by  %  inch, 
several  standardized  cylindrical  graduates  (1000  cc.  capacity)  graduated  in  10 
cc.,  a  half  dozen  pipettes  (5  cc.  capacity)  graduated  in  J^Q  cc.,  a  dozen  pipettes 
(i  cc.  capacity)  graduated  in  J^oo  cc.  (these  pipettes  should  discharge  between 
graduation  marks  and  should  not  be  graduated  to  the  tip),  several  graduated 
Luer  glass  syringes  (2  cc.  capacity),  several  lo-cc.  capacity  Luer  glass  syringes, 
100  Gm.  chemically  pure  sodium  chloride,  100  Gm.  chemically  pure  sodium 
citrate,  chloroform,  ether  and  non-absorbent  cotton. 

Needles  are  also  required.  Either  steel  or  iridium-platinum  needles  may 
be  used.  The  initial  cost  of  the  former  is  very  much  less  than  the  latter,  but 
eventually  the  less  corrosive  are  probably  the  cheaper.  The  iridium-platinum 
needles  are  also  the  nicer  to  work  with.  For  the  2-cc.  syringes,  needles  about 
i  J^  inches  long,  with  the  smallest  bore  through  which  blood  cells  can  be  passed, 
are  required.  The  needles  for  the  lo-cc.  syringes  are  used  for  withdrawing 
blood  from  the  veins  of  patients  and  from  the  hearts  of  rabbits  or  guinea-pigs. 
It  is  best  to  have  several  sizes  of  these,  the  most  useful  being  the  larger-size 
serum  needle. 

All  glassware  used  in  this  work  should  be  thoroughly  washed  in  several 
changes  of  distilled  water  and  be  free  of  acids  or  alkalies;  it  should  be  set  aside 
for  this  work  exclusively  and  thoroughly  washed  in  plain  water  immediately 
after  use.  For  some  complement  fixation  tests  it  is  essential  that  apparatus 
be  sterile.  It  is  a  good  practice  to  follow  in  all  these  tests  even  when  not  abso- 
lutely necessary,  and  it  is,  therefore,  recommended  that  all  containers — flasks, 
bottles,  test-tubes,  pipettes,  syringes  and  needles  be  sterilized  immediately  after 
use  and  kept  in  sterile  containers. 

In  our  own  laboratory  all  empty  glassware  is  sterilized  in  a  hot-air  sterilizer 
at  i4o°C.  for  2  hours.  Before  sterilization,  pipettes  are  placed  in  copper  con- 
tainers or  wrapped  in  paper;  Petri  dishes  and  syringes  are  enveloped  in  double 
paper  wrappers,  tubes  used  for  fixation  tests  are  wrapped  in  paper  in  packages 

226 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  227 

of  six,  all  other  tubes  are  plugged  with  cotton,  flasks  are  plugged  with  cotton, 
and  flasks  and  bottles  are  also  capped.  Normal  salt  and  sodium  citrate  solu- 
tions are  autoclaved  at  15  pounds  pressure  for  20  minutes  or  heated  20  minutes 
each  day  for  3  consecutive  days  in  a  steam  sterilizer.  Needles  are  kept  in  50 
per  cent,  alcohol.  They  are  flamed  just  before  use. 

The  form  of  test-tube  rack  is  a  matter  of  personal  selection;  the  author  pre- 
fers a  heavy,  rigid,  metal  rack  built  to  carry  three  tiers  of  12  tubes  each,  in  align- 
ment (Fig.  36). 

INCUBATOR 

Biochemical  reactions,  such  as  complement  fixation,  proceed  best  at  tem- 
peratures that  approximate  normal  body  temperature — 37°C. — consequently 
an  incubator  that  will  constantly  maintain  this  temperature  is  necessary. 

NORMAL  SALT  AND  SODIUM  CITRATE  SOLUTIONS 

When  red  cells  (corpuscles)  of  the  blood  are  removed  from  their  natural 
vehicle  (blood  serum)  and  placed  in  another  fluid,  their  normal  properties  are 
retained,  altered  or  destroyed  according  as  to  whether  the  fluid  in  which  they 
are  placed  is  isotonic,  hypertonic  or  hypotonic. 

If  red  cells  are  placed  in  water  they  immediately  burst,  liberate  their  hemo- 
globin and  dissolve;  if  they  are  placed  in  normal  salt  solution  they  can  remain 
viable  and  retain  all  their  normal  properties  for  days.  Red  cells,  separated 
from  their  native  serum,  are  required  in  the  Wassermann  and  other  Comple- 
ment Fixation  tests;  hence  normal  salt  solution  is  the  sole  vehicle  and  dilutent 
used  in  this  work. 

Normal  salt  solution  is  made  by  dissolving  8.5  Gm.  of  sodium  chloride  in 
1000  cc.  of  distilled  water.  To  preclude  undesirable  changes  normal  salt  solu- 
tion should  be  sterilized  as  soon  as  it  is  made  and  stored  in  sterile  containers 
having  a  capacity  of  not  more  than  200  cc. 

If  blood  is  withdrawn  from  an  animal  and  placed  in  a  container  it  will  clot 
in  a  few  minutes;  the  corpuscles  are  embedded  in  the  clot  and  cannot  be  extri- 
cated. Since  red  blood  cells  are  essential  in  making  complement  fixation  tests, 
it*  is  necessary  to  collect  the  blood  in  a  manner  that  will  prevent  coagulation. 
Sodium  citrate  precipitates  the  calcium  salts  of  the  blood  and  so  prevents  its 
coagulation;  hence  sodium  citrate  solution  is  used  in  collecting  blood  from  which 
to  obtain  red  cells  for  complement  fixation  tests. 

Sodium  citrate  solution  is  made  by  dissolving  10  Gm.  of  sodium  citrate  in 
1000  cc.  of  normal  salt  solution.  Sodium  citrate  solution  should  be  sterilized 
when  made  and  stored  in  sterile  containers. 

BLOOD  CELLS  AND  SENSITIZATION  OF  RABBITS 

After  procuring  the  necessary  apparatus  and  preparing  normal  salt  and 
sodium  citrate  solutions,  the  next  step  in  preparing  for  complement  fixation 
tests  is  the  sensitization  of  a  rabbit  against  red  blood  cells.  The  rabbit  must 
be  sensitized  to  the  same  kind  of  red  blood  cells  that  will  subsequently  be  used 


228  MEDICAL  BACTERIOLOGY 

in  performing  the  complement  fixation  tests.  Sheep's  red  blood  cells  and  human 
red  blood  cells  are  those  most  commonly  used.  So  far  as  the  value  and  end 
results  of  complement  fixation  tests  are  concerned  it  makes  no  difference  whether 
human  or  sheep  cells  are  used,  statements  to  the  contrary  notwithstanding.  If 
one  is  situated  where  sheep's  cells  can  be  procured  they  should  be  used,  under 
other  circumstances  human  cells  should  be  used.  If  one  has  access  to  a  sheep 
slaughter  house  it  is  most  convenient  and  economical  to  obtain  the  blood  there. 
Sheep  kept  in  the  laboratory  for  this  purpose  may  be  bled  every  week  or  10  days 
without  injury.  This  is  done  by  thrusting  a  strong,  heavy,  sharp,  hollow  needle 
into  the  jugular  vein  after  the  neck  has  been  shaved.  After  the  needle  is  with- 
drawn no  dressing  other  than  styptic  collodion  need  be  applied  to  the  wound. 
When  blood  is  withdrawn  from  a  man  for  the  purpose  of  obtaining  cells,  the 
most  prominent  superficial  vein  is  sought  for,  usually  at  the  elbow,  the  skin  over- 
lying it  is  washed  with  alcohol,  tincture  of  iodine  applied  for  i  minute  and  the 
area  again  washed  with  alcohol,  a  sterile  hollow  needle  is  then  thrust  into  the 
vein  and  the  blood  allowed  to  flow  from  the  needle. 

All  the  red  blood  cells  required  at  any  one  time  will  be  contained  in  50  cc.  of 
blood.  The  blood  is  collected  as  follows:  A  clean  sterile  glass  bottle  (200  cc. 
capacity)  having  a  wide  mouth  and  a  ground-glass  stopper  is  half  filled  with 
sodium  citrate  solution(Fig.  3;A);  blood  is  allowed  to  flow  into  the  bottle  until 
it  is  three-fourths  full  (Fig.  37C) — it  must  never  be  allowed  to  overflow — the 
stopper  is  inserted  and  the  bottle  shaken  to  thoroughly  mix  its  contents;  it  is 
at  once  transported  to  the  laboratory  and  kept  in  a  refrigerator  until  used.  The 
length  of  time  blood  so  collected  remains  fit  for  use  varies  from  i  day  to  several 
weeks,  hence  it  is  always  advisable  to  obtain  it  fresh  each  time  it  is  required. 

To  separate  the  red  blood  cells  the  citrated  blood  is  poured  into  i5-cc.  ca- 
pacity tubes  (Fig.  38A)  and  centrifugalized  until  all  the  cells  settle;  when  this  has 
taken  place  the  lower  portion  of  the  tubes  will  show  a  solid  red  opaque  mass 
(the  cells)  above  which  will  be  clear,  colorless,  transparent  fluid  (serum  and 
citrate  solution)  (Fig.  386).  The  supernatant  fluid  is  withdrawn  with  a  pipette, 
care  being  taken  not  to  disturb  the  cells  (Fig.  380,  D).  The  remaining  cells 
still  have  some  serum  and  citrate  solution  surrounding  them,  this  must  be  re- 
moved;.it  is  done  by  washing.  The  cells  are  washed  as  follows:  The  tubes  con- 
taining the  cells  are  filled  to  within  i  or  2  centimeters  of  the  top  with  normal 
salt  solution,  a  clean  thumb  is  placed  across  the  top  of  the  tube  to  confine  its 


EXPLANATORY  REMARKS  TO  FIG.  36. 

Ai,  A2  and  AS.     These  tubes  are  for  one  patient's  serum  under  examination. 

Bi,  B2  and  63.    These  tubes  are  for  a  second  patient's  serum  under  examination. 

Di,  D2  and  Da.  These  tubes  contain  the  known  syphilitic  patient's  serum  and  are,  therefore,  control 
tubes. 

Er,  E2  and  £3.    These  tubes  contain  non-syphilitic  patient's  serum  and  are,  therefore,  control  tubes. 

Ai,  Bi,  Di  and  Ei  contain  no  syphilitic  antigen,  therefore  these  tubes  should  show  an  illustrated  com- 
plete hemolysis. 

E2  and  £3  show  complete  hemolysis,  indicative  of  a  negative  reaction. 

D2  shows  no  hemolysis. 

D3  only  partial  hemolysis  indicative  of  a  weak  positive  reaction. 

B2  and  83  show  no  hemolysis.     A  strong  positive  reaction  indicative  of  syphilis. 

A2  and  A3  show  complete  hemolysis,  a  negative  reaction  not  indicative  of  syphilis. 

Ji  is  a  complement  control  tube.  Contains  nothing  but  guinea-pig  serum  and  red  cells.  This  tube 
should  show  no  hemolysis  as  indicated. 


J2.     Hemolytic  system  control,  complete  hemolysis. 
]2 


Syphilitic  antigen  control.     Complete  hemolysis  (see  pages  243  and  247). 


I 


f 


B 


f 


Fig.  38. 

A,  Citrated  blood  as  poured  into  centrifuge  tube.  B,  Cells  precipitated  to  the  bottom  of 
the  tube,  clear  serum  and  citrate  solution  above.  Result  of  centrifugalizing  A.  C,  Removal 
of  supernatant  fluid.  D,  Test  tube  containing  red  cells  after  supernatant  fluid  has  been 
removed. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  22Q 

contents  and  the  tube  is  then  inverted  and  shaken  a  sufficient  number  of  time 
to  evenly  distribute  the  cells  throughout  the  fluid;  the  tubes  are  again  centrifu- 
gahzed  until  all  the  cells  settle  to  the  bottom;  the  salt  solution  is  pipetted  off 
This  washing  is  repeated  twice.     After  the  third  washing  the  cells  are  ready  to 
be  injected  into  the  rabbit. 

Only  large  healthy  rabbits  should  be  used.     With  a  pair  of  scissors  the  hair 
is  removed  so  as  to  expose  one  of  the  veins  on  the  margin  of  the  ear.     Half  a 


A 


FIG.  37. 

A,  Sterilized  glass  bottle  with  wide  mouth  and  ground  glass  stopper,  half  filled  with 
sodium  citrate  solution.     B,  Blood  from  sheep  flowing  into  bottle   (C)  until  the  bottle  is 

three-fourths  full. 


cubic  centimeter  of  red  blood  cells  is  poured  into  a  small  (2  cc.  capacity)  Luer 

syringe,  and  with  the  finest  needle  through  which  the  cells  will  pass,  they  are 

injected  into  the  vein  (Fig.  39). 

Twenty-four  hours  after  the  first  injection,  in  exactly  the  same  manner,  the 

second  is  given,  the  quantity  of  red  blood  cells  injected  being  i  cc. 

Twenty-four  hours  after  the  second  injection  a  third  is  given — quant  it  \ 
Twenty-four  hours  after  the  third  injection  the  last  one  is  given— quantity 

3  cc. 


230 


MEDICAL  BACTERIOLOGY 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS 


231 


The  rabbit  is  then  allowed  to  rest  for  10  days,  during  which  time  he  develops 
specific  antibodies  for  red  blood  cells,  such  as  were  injected.  When  sensitized 
the  rabbit  is  anesthetized  with  ether  and  bled  by  one  of  the  following  methods : 

First. — By  touch  locate  the  heart,  thrust  a  sterile  needle  into  it  and  with  a 


FIG.  40. — WITHDRAWING  BLOOD  FROM  THE  HEART  OF  AN  ANESTHETIZED  RABBIT. 

lo-cc.  Luer  syringe  withdraw  30  cc.  of  blood  (Fig.  40).  Transfer  the  blood  from 
the  syringe  into  a  sterile  Petri  dish.  Inject  30  cc.  of  warm  normal  salt  solution 
into  the  rabbit's  peritoneal  cavity  (Fig.  41). 

Second. — Incise  the  skin  at  the  tip  of  the  sternum  with  a  pair  of  scissors,  one 
blade  of  which  has  a  blunt  end  and  the  other  a  pointed  end.  Elevate  the  skin 
and  thrust  the  blunt  end  of  the  scissors  into  the  incision,  pushing  it  in  the  median 


232 


MEDICAL  BACTERIOLOGY 


X. 


,  - 


I 


\ 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS 


line  toward  the  rabbit's  head.  The  scissors  must  be  kept  between  the  skin  and 
superficial  fascia.  When  the  skin  has  been  divided  from  the  sternum  to  the 
chin,  it  is  rapidly  dissected  back  until  the  anterior  half  of  the  rabbit's  neck  is 
denuded.  The  rabbit  is  then  grasped  by  the  hind  legs,  held  vertically  over  a 
Petri  dish  so  that  the  blood  will  flow  directly  into  it  when  the  jugular  veins  are 
cut.  Care  must  be  taken  to  avoid  opening  the  trachea  or  esophagus  when  in- 
cising the  jugular  veins  (Fig.  42). 


FIG.  42. — BLOOD  FLOWING  FROM  INCISED  JUGULAR  VEIN  OF  RABBIT  INTO  PETRI  DISH. 

The  Petri  dishes  containing  the  blood  are  covered  and  placed  in  a  refrigerator 
for  from  12  to  24  hours.  At  the  expiration  of  this  time  the  Petri  dishes  are  re- 
moved from  the  refrigerator  and  the  serum  (fluid)  is  transferred  to  tubes  and 
centrifugalized  until  perfectly  clear  and  free  of  suspended  matter.  It  is  then 
transferred  to  clean  tubes  which  are  placed  in  a  water  bath  at  a  temperature 
of  55°C.  or  56°C.  for  }/%  hour.  When  removed  from  the  water  bath  about  i  per 
cent,  of  chloroform  is  added  to  the  serum  and  it  is  then  put  in  i-cc.  capacity 
vials  which  are  tightly  stoppered  with  rubber  plugs,  then  stored  in  a  refrigerator. 


234  MEDICAL  BACTERIOLOGY 

DILUTION  OF  BLOOD  CELLS 

In  performing  complement  fixation  tests,  as  hereafter  described,  red  blood 
cells  are  required.  They  are  procured  as  described  in  the  preceding  chapter 
and  are  washed  twice.  After  the  second  washing  a  5  per  cent,  suspension  is 
made  by  mixing  each  cubic  centimeter  of  red  blood  cells  with  19  cc.  of  normal 
salt  solution.  When  red  blood  cells  are  to  be  added  to  a  tube,  invariably,  i  cc. 
of  a  5  per  cent,  suspension  is  the  amount  added.  To  avoid  cumbersome  ex- 
pression, the  term  "red  cells"  will  hereafter  be  used  in  this  volume  to  designate 
i  cc.  of  a  5  per  cent,  suspension  of  red  blood  cells  in  normal  salt  solution. 

COMPLEMENT  AND  AMBOCEPTOR 

Experiment  No.  1. — Take  a  normal  healthy  rabbit  that  has  been  subjected 
to  no  treatment,  obtain  some  blood  from  it,  place  this  blood  in  a  refrigerator 
until  it  clots  and  serum  separates  from  it. 

Into  a  number  of  test-tubes  put  various  amounts  of  this  serum — from  o.i 
to  2.0  cc.,  add  red  cells  to  each  tube.  Shake  the  tubes  to  mix  their  contents. 
Note  their  appearance.  Place  the  tubes  in  an  incubator  for  i  or  2  hours,  then 
inspect  them  and  transfer  them  to  a  refrigerator;  several  hours  later  make  a 
final  inspection. 

Before  they  were  placed  in  the  incubator  these  tubes  showed  a  homogeneous, 
bright  red,  opaque  fluid  and  no  precipitate.  At  the  end  of  incubation  the  ex- 
treme upper  zone  of  the  fluid  was  clear,  colorless  and  transparent,  on  the  bot- 
tom of  the  tubes  there  was  a  slight,  dark  red  precipitate  and  between  these  two 
the  fluid  was  still  bright  red  and  opaque.  At  the  final  inspection  a  solid,  dark 
red,  opaque  sediment,  about  J^  inch  in  depth,  covered  the  bottom  of  the  tubes; 
above  this  the  fluid  was  clear,  colorless  and  transparent — like  water;  as  B2, 
Fig.  36. 

When  the  serum  and  cells  were  mixed  in  the  tubes  by  shaking,  the  cells  were 
evenly  distributed  throughout  the  fluid,  hence  the  homogeneous,  bright  red, 
opaque  appearance  and  absence  of  sediment.  When  these  tubes  had  stood  at 
rest  for  an  hour  or  two  in  the  incubator  the  cells  gradually  precipitated;  the  pre- 
cipitation being  only  partial,  but  a  slight  zone  at  the  top  of  the  fluid  was  entirely 
free  of  cells,  hence  only  this  slight  zone  was  clear,  colorless,  and  transparent 
like  water,  and  only  a  slight  sediment  was  visible  at  the  bottom  of  the  tubes. 

When  the  tubes  were  finally  inspected,  after  standing  at  rest  in  the  incubator 
and  ice  box  for  from  4  to  6  hours,  sufficient  time  had  elapsed  for  precipitation  to 
be  complete,  in  which  case  all  the  cells  were  on  the  bottom  of  the  tube;  all  the 
fluid  being  free  of  cells  consequently  had  the  appearance  of  water. 

The  above  experiment  furnishes  visible  evidence  that  fresh,  unheated  serum 
from  a  normal  untreated  rabbit  has  no  effect  on  red  blood  cells — whether  they 
be  obtained  from  a  man,  sheep  or  ox.*  This  is  referred  to  as  no  hemolytic 
effect  or  no  hemolysis. 

*  Rabbit  serum  may  occasionally  contain  natural  Hemolysin  for  foreign  red  cells. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  235 

Experiment  No.  2. — Sensitize  a  rabbit  to  red  blood  cells  as  described  in  the 
preceding  chapter;  obtain  some  of  its  blood,  allow  this  blood  to  stand  in  a  re- 
frigerator until  serum  separates;  collect  the  serum  and  put  various  amounts  of 
it  in  test-tubes — from  o.ooi  to  i.o  cc.,  add  red  cells  to  each  tube;  shake  the  tubes 
to  mix  their  contents;  inspect  them,  their  appearance  will  be  identical  to  that 
observed  prior  to  incubation  in  experiment  No.  i. 

Place  the  tubes  in  an  incubator  for  i  to  2  hours,  then  remove  and  again  in- 
spect; there  will  be  a  radical  change  in  their  appearance.  Some  of  the  tubes 
containing  the  larger  amounts  of  serum  will  show  a  clear,  transparent,  bright 
red  solution,  the  same  in  appearance  throughout  and  there  will  be  no  sediment 
whatever  in  these  tubes.  Some  of  the  tubes  containing  the  smallest  amounts 
of  serum  will  present  the  same  appearance  as  was  observed  at  the  end  of  incu- 
bation in  experiment  No.  i.  Between  these  two  groups  of  tubes  will  be  a  num- 
ber in  which,  as  the  amount  of  serum  increases,  the  amount  of  opaque  fluid 
diminishes,  the  amount  of  sediment  diminishes  and  the  amount  of  clear  trans- 
parent red  fluid  increases. 

When  tubes  to  which  red  blood  cells  have  been  added  show  a  clear,  trans- 
parent red  fluid  and  no  sediment,  no  matter  how  long  the  tubes  stand  at  rest, 
it  is  evidence  that  the  red  blood  cells  have  been  disintegrated  and  their  hemo- 
globin gone  into  solution;  this  change  is  termed  hemolysis,  and  the  tubes  have 
the  same  appearance  as  Ai,  Fig.  36. 

Experiment  No.  2  shows  that  injection  of  red  blood  cells,  from  another 
species,  into  rabbits,  stimulates  the  production  of  antibodies ;  that  these  anti- 
bodies are  present  in  the  blood  serum  of  such  rabbits;  that  when  this  serum  in 
the  fresh  state  and  proper  amount  is  added  to  red  cells  such  as  were  used  to 
stimulate  the  production  of  antibodies,  it  will  destroy  or  hemolize  the  red  cells. 

Experiment  No.  3. — Repeat  Experiment  No.  2,  all  factors  being  the  same 
except  that  the  rabbit  serum  be  heated  in  a  water  bath  at  55°C.  or  56°C.  for 
3^  hour  immediately  before  mixing  with  the  red  cells. 

There  will  be  no  hemolysis.  This- demonstrates  that  heating  the  serum  con- 
taining hemolytic  antibodies  so  alters  the  antibodies  as  to  deprive  them  of  their 
hemolytic  power. 

Experiment  No.  4. — Repeat  Experiment  No.  3,  all  factors  being  the  same,  ex- 
cept that  in  addition  to  placing  various  amounts  of  heated-sensitized-rabbit  serum 
and  red  cells  in  every  tube,  o.i  cc.  of  fresh  unheated  serum,  derived  from  an 
untreated,  unsensitized  normal  rabbit,  is  added  to  each  tube  before  incubation. 

When  these  tubes  are  examined  after  incubation  hemolysis  will  be  observed 
the  same  as  in  Experiment  No.  2 — evidence  that  the  antibodies,  present  in  the 
serum  of  rabbits  sensitized  to  red  blood  cells,  are  composed  of  two  parts — one 
which  is  destroyed  by  heating  to  55°C.  for  %  hour,  called  complement;  the  other 
which  is  not  injured,  altered  or  destroyed  by  so  heating,  called  amboceptor. 

The  experiment  also  shows  that  complement  is  a  native  constituent  of  all 
rabbits,  untreated  and  unsensitized  as  well  as  those  that  have  been  treated  or 
sensitized;  and  that  the  complement  in  one  rabbit's  serum  can  unite  with  the 
amboceptors  of  another  rabbit's  serum. 


•2$6  MEDICAL  BACTERIOLOGY 

By  varying  and  extending  these  experiments  it  can  be  demonstrated  that 
complement  is  present  in  the  blood  serum  of  all  warm-blooded  animals  through- 
out life.  The  amount  of  complement  present  per  cubic  centimeter  in  the  blood 
serum  is  about  the  same  in  any  given  animal  at  all  times.  The  amount  of 
complement  per  cubic  centimeter  in  the  serum  of  one  animal  is  nearly  the  same 
-in  all  other  animals  of  the  same  species,  but  variations  do  occur.  The  comple- 
ment present  in  the  serum  of  an  animal  can  unite  with  any  amboceptor  present 
in  the  serum  of  that  animal  or  any  other  animal  of  the  same  species.  Comple- 
ment of  identical  nature  is  possessed  by  several  species  and  is  therefore  capable 
of  uniting  with  amboceptors  found  in  any  of  them.  The  complement  present 
in  the  serum  of  guinea-pigs,  rabbits  and  man  is  practically  the  same,  therefore, 
guinea-pig  complement  can  unite  with  amboceptors  present  in  human  serum, 
and  those  in  rabbit  serum.  Complement  is  at  once  destroyed  by  heating  serum 
,to  55°C.  for  J^  hour;  it  gradually  leaves  the  serum  after  the  serum  is  withdrawn 
From  an  animal,  even  though  the  serum  is  not  heated.  If  serum  is  allowed  to 
stand  at  room  temperature  it  loses  its  complement  in  about  24  hours  or  less, 
[f  serum  is  placed  in  a  refrigerator  immediately  after  taken  from  an  animal  it 
detains  its  complement  for  from  24  to  72  hours.  If  serum  is  frozen  immediately 
after  taken  from  an  animal  it  retains  its  complement  for  a  number  of  days. 

The  complement-amboceptor  experiments  demonstrate  that  a  given  amount 
of  complement  will  unite  with  a  definite  amount  of  amboceptor  and  no  more. 

Amboceptor  is  specific  in  action — it  will  only  attack  the  substance  that  stim- 
ulated its  production. 

Amboceptor  only  unites  with  complement  in  the  presence  of  the  substance 
which  stimulated  the  production  of  the  amboceptor.  When  complement  unites 
with  an  amboceptor  it  can  never  leave  that  amboceptor  to  unite  with  another 
amboceptor.  The  amount  of  amboceptor  per  cubic  centimeter  in  the  serum  of 
an  animal  depends  upon  the  method  of  inoculation  with  the  substance  that  stimu- 
lates amboceptor  production,  upon  the  size  and  number  of  inoculations  and 
the  interval  between  the  time  of  inoculation  and  the  time  when  serum  is 
examined. 

The  amount  of  amboceptor  per  cubic  centimeter  in  the  serum  of  any  given 
animal  varies  from  time  to  time,  usually  growing  less  as  the  animal  ages. 

The  amount  of  amboceptor  per  cubic  centimeter  in  the  serum  of  different 
animals  of  the  same  species  subjected  to  identical  inoculations  is  subject  to 
wide  variations. 

When  serum  containing  amboceptor  is  taken  from  an  animal  under  aseptic 
precautions,  placed  in  hermetically  sealed  containers  and  immediately  stored  in 
a  refrigerator,  the  amount  of  amboceptor  per  cubic  centimeter  will  remain  almost 
constant,  decreasing  only  a  small  fraction  of  a  per  cent,  per  month,  for  many 
months. 

The  addition  of  chloroform  to  serum  to  prevent  bacterial  growth  has  no 
effect  on  its  amboceptor  content.  Amboceptor  is  not  affected  by  heating  to 
55°C.  for  J£  hour;  a  temperature  of  about  6o°C.  or  7o°C.  will  destroy  it. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  237 

STANDARDIZATION  OF  AMBOCEPTOR 

When  performing  complement  fixation  tests  one  adds  i  cc.  of  a  5  per  cent, 
suspension  of  red  blood  cells  to  every  tube,  an  amount  of  rabbit  serum  that  con- 
tains sufficient  amboceptor  to  insure  complete  hemolysis  of  these  cells,  with 
the  amount  of  complement  placed  in  the  tubes,  must  also  be  added.  As  already 
stated  rabbits  treated  in  an  identical  manner  give  serum  which  varies  in  ambo- 
ceptor content  of  immunized  rabbit  serum  when  one  procures  it.  To  do  this 
a  unit  of  measurement  must  be  established  and  for  this  purpose  the  following 
rule  has  been  established: 

A  unit  of  amboceptor  is  the  smallest  amount  that  will  cause  complete  hem- 
olysis of  i  cc.  of  a  5  per  cent,  suspension  of  red  blood  cells,  together  with  i  cc. 
of  a  10  per  cent,  solution  of  fresh  guinea-pig  serum  (complement),  when  incu- 
bated for  i  hour  at  37°C. 

Rabbit  serum  is  not  acceptable  unless  a  unit  of  amboceptor  is  contained  in 
less  than  o.oi  cc.  of  serum.     The  required  accuracy  of  measurement  cannot  be', 
accomplished,  under  ordinary  circumstances,  if  one  attempts  to  discharge  from 
a  graduated  pipette  less  than  o.i  cc.  of  fluid — therefore,  the  first  step  in  prepar- 
ing amboceptor  for  standardization  is  to  make  a  i  per  cent,  solution  of  the  rab- 
bit serum  in  normal  salt  solution.     To  9.9  cc.  of  normal  salt  solution  add  o.i  cc. 
of  rabbit  serum,  mix  thoroughly.  • 

Take  a  test-tube  rack  built  to  hold  1 2  tubes  and  place  a  tube  in  each  of  the 
first  10  holes  and  in  the  twelfth  hole. 

Put  o.i  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  first  tube. 

Put  0.2  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  second  tube. 

Put  0.3  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  third  tube. 

Put  0.4  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  fourth  tube. 

Put  0.5  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  fifth  tube. 

Put  0.6  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  sixth  tube. 

Put  0.7  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  seventh  tube. 

Put  0.8  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  eighth  tube. 

Put  0.9  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  ninth  tube 

Put  i.o  cc.  of  the  i  per  cent,  solution  of  rabbit  serum  in  the  tenth  tube. 

Do  not  put  any  rabbit  serum  in  the  last  tube. 

Put  i  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  every  tube. 

Put  i  cc.  of  the  5  per  cent,  suspension  of  red  blood  cells  in  every  tube. 

Shake  the  tubes  to  thoroughly  mix  their  contents  and  place  in  incubator. 

At  the  end  of  an  hour  remove  and  inspect  the  tubes,  the  appearance  may  be 
as  follows: 

The  first  one,  two  or  three  tubes  may  show  little  or  no  hemolysis;  the  next 
two  or  three  tubes  progressively  increasing  amounts  of  hemolysis  and  the 
rest,  up  to  and  including  the  tenth  tube,  complete  hemolysis,  the  last  tube  no 
hemolysis. 

Such  an  observation  would  establish  the  following:  Absence  of  anything 
other  than  complement  in  the  guinea-pig  serum  capable  of  hemolizing  red  cells 
as  evidenced  by  the  last  tube. 


238  MEDICAL  BACTERIOLOGY 

The  presence  of  complement  in  the  guinea-pig  serum,  shown  by  the  occur- 
rence of  complete  hemolysis  in  the  tubes  containing  a  unit  or  more  of  amboceptor; 
that  a  sufficient  number  of  tubes  were  run  to  determine  the  unit  of  amboceptor, 
because  in  addition  to  the  tubes  showing  complete  hemolysis  there  were  others, 
containing  smaller  amounts  of  rabbit  serum,  in  which  hemolysis  was  incomplete, 
slight,  or  did  not  occur. 

When  a  sensitized  rabbit  is  bled  from  10  to  20  cc.  of  serum  is  obtained,  an 
amount  sufficient  for  many  hundreds  of  complement  fixation  tests.  As  the 
amboceptor  content  of  this  serum  will  remain  practically  constant,  once  the 
unit  of  a  particular  rabbit's  serum  has  been  determined,  that  quantity  is  taken 
as  the  measure  with  which  to  determine  the  unit  of  complement  prior  to  the 
performance  of  each  subsequent  complement  fixation  test. 

COMPLEMENT 

Fresh  unheated  guinea-pig  serum  is  ordinarily  used  to  supply  the  comple- 
ment for  complement  fixation  tests.  Guinea-pig  serum  is  selected  because  it 
contains  complement  capable  of  uniting  with  amboceptors  of  either  rabbit  or 
human  serum;  because  the  average  normal  guinea-pig  serum  will  not  hemolize 
human  or  sheep  red  blood  cells;  guinea-pigs  are  comparatively  easy  to  obtain, 
keep,  handle  and  bleed. 

The  method  of  bleeding  guinea-pigs  is  the  same  as  described  on  page  231 
for  bleeding  sensitized  rabbits.  After  some  practice  one  can  save  time  and 
labor  by  withdrawing  the  blood  from  the  pig's  heart  with  a  syringe.  There 
are  other  advantages  in  so  obtaining  the  blood:  (i)  When  not  more  than  2  cc. 
of  serum  is  required  the  withdrawal  of  the  amount  of  blood  necessary  to  yield 
the  desired  serum  (about  5  cc.)  can  usually  be  accomplished  without  permanent 
injury  to  the  pig.  (2)  For  reasons  to  be  explained  later,  it  is  better  to  use  a 
mixture  of  the  serum  from  several  pigs  rather  than  the  serum  of  a  single  pig. 
(3)  When  not  more  than  5  cc.  of  blood  is  withdrawn  from  a  pig,  if  an  equal 
amount  of  normal  salt  solution  is  injected  into  the  peritoneal  cavity,  the  pig 
usually  survives  and  several  weeks  later  may  be  bled  again.  Some  pigs  survive 
as  many  as  10  bleedings. 

When  there  is  no  particular  hurry  it  is  best  to  bleed  pigs  into  Petri  dishes, 
place  the  dishes  in  a  refrigerator  over  night  and  collect  the  serum  and  make  the 
complement  fixation  tests  the  following  morning.  In  this  way  the  maximum 
amount  of  serum  is  obtained  with  the  least  labor.  To  obtain  the  serum  rapicUy, 
bleed  the  pigs  into  centrifuge  tubes,  stir  the  blood  with  a  glass  rod  and  centri- 
fugalize  it  at  high  speed  for  5  or  10  minutes,  the  clear  serum  will  rise  to  the 
top  and  can  be  at  once  pipetted  off  and  used. 

While  the  amount  of  complement  per  cc.  in  the  serum  of  a  guinea-pig  is 
nearly  constant,  individual  exceptions  to  the  rule  are  met.  The  variations  will 
be  slightest  when  serum  from  several  pigs  is  mixed.  Although  the  serum  of 
most  guinea-pigs  will  not  hemolize  sheep  or  human  red  blood  cells,  occasionally 
a  serum  is  obtained  that  does.  For  these  reasons  it  is  advisable  to  standardize 
complement  before  using  it.  The  method  of  standardization  is  as  follows: 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  239 

Take  a  test-tube  rack  built  to  carry  1 2  tubes  in  a  row  and  place  a  tube  in 
each  of  the  first  10  holes  and  one  in  the  twelfth. 

Make  a  10  per  cent,  solution  of  fresh  guinea-pig  serum  in  normal  salt  solu- 
tion by  mixing  i  cc.  of  serum  with  9  cc.  of  salt  solution. 

Put  0.3  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  first  tube. 

Put  0.4  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  second  tube. 

Put  0.5  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  third  tube. 

Put  0.6  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  fourth  tube. 

Put  0.7  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  fifth  tube. 

Put  0.8  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  sixth  tube. 

Put  0.9  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  seventh 
tube. 

Put  i.o  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  eighth  tube. 

Put  i.i  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  ninth  tube. 

Put  1.2  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  tenth  tube. 

Put  i  cc.  of  the  10  per  cent,  solution  of  guinea-pig  serum  in  the  last  tube. 

Put  one  unit  of  amboceptor  in  every  tube  except  the  last.  Do  not  put  any 
amboceptor  in  the  last  tube. 

Put  i  cc.  of  5  per  cent,  suspension  of  red  blood  cells  in  every  tube. 

Shake  the  tubes  to  mix  their  contents  and  place  in  incubator. 

At  the  end  of  an  hour  remove  and  inspect  the  tubes. 

The  last  tube  contains  guinea-pig  serum  and  blood  cells  only;  if  any  hemoly- 
sis  occurs  in  this  tube  it  indicates  that  the  guinea-pig  serum  is  of  itself  destruc- 
tive to  blood  cells  and  hence  cannot  be  used  in  making  complement  fixation  tests. 
The  last  tube  should  show  no  hemolysis. 

Having  learned  from  inspection  of  the  last  tube  that  the  serum  is  accept- 
able, provided  it  contains  complement,  the  other  tubes  are  then  inspected  to 
detect  the  presence  and  quantity  of  complement. 

A  unit  of  complement  is  the  smallest  amount  that  will  cause  complete 
hemolysis  of  i  cc.  of  a  5  per  cent,  suspension  of  red  cells,  together  with  one 
unit  amboceptor,  when  incubated  at  37°C.  for  i  hour. 

THE  WASSERMANN  TEST 

Just  as  normal  untreated  rabbits  have  no  amboceptors  in  their  blood  serum 
which  can  act  on  red  blood  cells,  normal  humans  who  have  not  been  infected 
with  syphilis  have  no  amboceptors  in  their  blood  serum  which  can  act  on  syphi- 
litic antigen. 

Just  as  rabbits  that  have  been  injected  with  red  cells  do  have  amboceptors 
in  their  serum  which  can  act  on  red  blood  cells,  humans  who  have  been  infected 
with  syphilis  do  have  amboceptors  in  their  serum  which  can  act  on  syphilitic 
antigen. 

The  Wassermann  test  is  a  test  to  determine  the  presence  or  absence  of  syphi- 
litic amboceptors  in  a  given  individual's  serum.  For  this  purpose  five  sub- 
stances are  necessary: 


240  MEDICAL  BACTERIOLOGY 

1 .  Syphilitic  Antigen : 

Contains  no  amboceptor,  if  negative. 

2.  Patient's  Serum :  c     .....  \^^\ 

(  Syphilitic  amboceptor,  if  positive. 

.—s/'VVN— 

3.  Complement  (guinea-pigs): 

4.  Amboceptor  (immunized  rabbits) : 

5.  Red  Blood  Cells : 

There  are  here  four  known  substances  with  which  to  determine  the  presence 
or  absence  of  a  fifth,  namely,  syphilitic  amboceptor  = 

It  is  known  that  complement  will  join  with  amboceptor  when  that  ambocep- 
tor's  antigen  is  present;  the  complement  is  then  fixed  and  can  never  join  any 
other  amboceptor.  Hence,  if  the  serum  of  a  syphilitic  person  is  placed  in  a  tube 
with  complement  and  syphilitic  antigen  and  incubated  for  an  hour  the  com- 
plement is  fixed.  There  is  no  free  complement  in  the  tube. 


U 


Incubated  at  37°C.  for  i  hour. 
Complement  fixation. 


If  the  serum  of  a  non-syphilitic  person  is  placed  in  a  tube  with  complement 
and  syphilitic  antigen  and  incubated  the  complement  is  not  fixed — the  comple- 
ment remains  free  and  unaltered. 

Incubated  at  37°C.  for  i  hour. 


No  Complement  Fixation. 

As  placed  in  the  tubes  the  complement,  human  serum,  syphilitic  antigen 
mixture  is  a  clear,  colorless  fluid,  almost  like  water  in  appearance.  No  change 
in  its  appearance  occurs  when  complement  is  fixed,  therefore,  it  is  necessary  to 
add  something  to  these  tubes  that  will  indicate  whether  the  complement  re- 
mained free  or  was  fixed.  For  this  purpose,  after  the  complement,  human 
serum,  syphilitic  antigen  mixture  has  been  incubated  for  i  hour,  red  blood  cells 
and  the  inactivated  serum  of  a  rabbit  sensitized  against  such  cells  are  added. 


WASSERMANN  AND   OTHER   COMPLEMENT  FIXATION  TESTS  241 

And  after  shaking,  the  tubes  are  incubated  a  second  time  for  i  hour,  then 
inspected. 

If  the  complement  was  fixed  during  the  first  hour  of  incubation,  then  there 
was  no  free  complement  in  the  tube  when  the  rabbit  serum  and  red  cells  were 
added;  since  amboceptor  alone  cannot  destroy  antigen,  the  red  cells  remain 
unaltered,  they  precipitate  to  the  bottom  of  the  tube,  and  fluid  above  appears 
as  water,  there  is  no  hemolysis.  Such  is  the  end  appearance  of  a  positive 
Wassermann  test,  indicative  of  syphilis. 


U 


Incubated 

i 
hour 


add 


Again  incubate 

i  hour 
no  hemolysis. 


If  the  complement  was  not  fixed,  remained  free,  during  the  first  hour  of 
incubation,  then  there  was  free  complement  in  the  tube  when  the  rabbit  serum 
and  red  cells  were  added.  Since  complement  will  join  with  amboceptor  when 
that  amboceptor's  antigen  is  present  and  thereby  form  an  antibody  that  will 
destroy  the  antigen,  the  red  cells  would  be  destroyed,  the  hemoglobin  dissolved, 
the  tubes  show  no  sediment,  but  a  clear,  transparent  red  fluid  hemolysis. 
Such  is  the  end  appearance  of  a  negative  Wassermann  test — not  indicative  of 
syphilis. 


U 

A 


Incubated 


hour. 


U 


add 


P   Again  incubate 
— J  i  hour 


hemolysis 


PATIENTS'  SERUM  FOR  THE  WASSERMANN  TEST 


Only  a  fraction  of  a  cc.  of  serum  is  required  for  a  Wassermann  test,  an 
amount  that  can  be  obtained  from  i,  2,  3  cc.  of  blood, but  in  all  cases  it  is  foolish 
to  withdraw  so  small  an  amount.  Frequently  it  is  desirable  to  make  the  test 
in  duplicate  or  triplicate;  it  is  well  to  have  an  excess  of  serum  that  may  be  kept 
in  storage  in  case  one's  findings  are  questioned,  so  it  can  be  submitted  to  several 
independent  workers  for  reexamination.  It  is  necessary  to  have  known  nega- 
tive and  positive  sera  to  control  tests  and  they  can  only  be  accumulated  by  ob- 
taining from  patients  more  serum  than  is  required  for  a  single  examination. 
No  matter  how  skillful  the  operator,  one  does  not  always  withdraw  the  full 
amount  contemplated  before  operation.  For  these  reasons  it  is  best  to  with- 
draw from  5  to  10  cc.  of  blood.  This  blood  may  be  obtained  in  several  ways: 

First. — Asepticize  a  finger,  incise  it  and  let  the  blood  flow  into  a  test-tube. 

Second. — Asepticize  an  area  over  the  most  prominent  vein  of  the  forearm, 
thrust  a  needle  into  it  and  let  the  blood  flow  from  the  needle  into  a  test-tube. 

Third. — Have  a  sterile  syringe  attached  to  the  needle  that  is  thrust  into  the 
vein,  withdraw  the  blood  with  the  syringe  and  empty  the  syringe  into  a  test-tube. 


242  MEDICAL  BACTERIOLOGY 

The  third  method  is  preferable  because  it  is  least  dangerous  to  the  patient 
and  least  painful.  It  does  not  require  as  large  a  needle  as  the  second  method 
and  precludes  soiling  the  clothes  with  blood  and  protects  the  withdrawn  blood 
from  contamination. 

The  first  method  is  that  of  necessity  and  should  never  be  resorted  to  when  it 
is  possible  to  obtain  the  blood  by  vein  puncture. 

However  the  blood  is  obtained  the  procedure  must  be  made  under  aseptic 
conditions  and  guarded  with  the  usual  surgical  precautions. 

The  blood  is  collected  in  a  sterile  test-tube  at  least  ^  inch  in  diameter.  If 
this  tube  is  slanted  as  soon  as  the  blood  enters  it,  and  allowed  to  remain  at  rest 
until  the  blood  coagulates,  then  placed  upright,  clear  serum  will  separate  in 
several  hours  and  can  then  be  poured  into  another  tube,  inactivated  and  tested. 
If  the  tube  must  be  kept  upright  from  the  time  the  blood  enters  it  the  serum  is 
apt  to  be  cloudy  and  should  the  interior  of  the  tube  be  rough,  the  diameter  less 
than  J^  inch,  serum  may  not  separate  from  the  clot  until  it  is  stirred  with  a  glass 
rod,  then  it  is  always  cloudy.  When  the  serum  is  cloudy,  has  suspended  matter 
in  it,  it  must  be  centrifugalized  until  perfectly  clear  and  free  of  suspended  matter 
before  testing.  As  a  result  of  forceful  expulsion  of  blood  from  a  syringe  into 
a  tube,  agitation  during  transportation  or  stirring  with  a  glass  rod,  blood  cells 
are  ruptured  and  their  hemoglobin  tints  the  serum  red,  this  discoloration  of  the 
serum  cannot  be  removed,  nor  is  there  any  need  to;  it  does  not  affect  the  test. 
Serum  that  is  sterile  may  be  kept  for  weeks  or  months  and  still  give  the  same 
reaction  as  when  recently  removed,  but,  for  reasons  that  will  be  later  explained, 
it  is  best  to  make  the  test  within  48  hours  after  removal  of  the  serum  from  the 
patient. 

Occasionally,  it  is  desirable  to  make  a  test  as  soon  as  possible,  then  the  blood 
is  collected  in  a  centrifuge  tube,  gently  stirred  with  a  glass  rod  and  centrifugal- 
ized at  high  speed  for  5  minutes ;  in  that  time  clear  serum  will  collect  at  the  top 
of  the  tube  and  can  be  at  once  removed,  inactivated  and  tested. 

The  experience  of  the  vast  majority  of  serologists  shows  it  is  best  to  inacti- 
vate the  patient's  serum.  This  deprives  it  of  complement.  The  serum  should 
be  inactivated  immediately  before  testing  and  control  sera  should  be  inactivated 
every  time  they  are  used,  regardless  of  how  often  that  may  be. 

Sera  are  inactivated  by  placing  the  tubes  containing  them  in  a  water  bath 
at  55°  or  56°C.  for  J^  hour.  It  will  not  matter  if  they  are  heated  a  few  minutes 
more,  but  it  is  imperative  that  the  temperature  is  maintained  at  55°C.  or  56°C. 

Just  as  different  rabbits  injected  with  red  blood  cells  have  different  amounts 
of  amboceptor  in  their  sera,  different  individuals  infected  with  syphilis  have 
different  amounts  of  amboceptor  in  their  sera.  Wassermann,  Neisser,  Buchner, 
Morgenroth,  Ehrlich  and  others  discovered  by  experimentation  that  from  0.20 
to  0.02  cc.  of  patient's  serum  might  be  used  to  make  a  complement  fixation  test 
for  syphilis. 

For  reasons  that  will  be  discussed  later,  o.i  cc.  of  patient's  serum  seems  the 
ideal  quantity  to  use,  and  in  the  Wassermann  test  as  described  in  this  book  o.i 
cc.  of  the  patient's  serum  is  the  amount  always  employed. 


WASSERMANN   AND    OTHER   COMPLEMENT    FIXATION   TESTS  243 

ANTIGEN  FOR  THE  WASSERMANN  TEST 

Remembering  that  amboceptor  does  not  fix  complement  unless  the  ambo- 
ceptor's  antigen  is  present,  it  is  obvious  that  a  syphilitic  patient's  serum, 
containing  syphilitic  amboceptors,  cannot  fix  complement  unless  syphilitic 
antigen  is  present.  Amboceptors  are  specific;  they  only  act  on  the  antigens 
that  stimulated  their  production.  Antigens  are  specific;  they  will  only  facil- 
itate complement  fixation  when  they  act  with  their  own  amboceptors. 

A  priori,  it  would  seem  that  the  amboceptors  of  syphilis  would  be  those 
formed  to  destroy  the  treponema  pallidum,  that  they  would  be  specific  for  the 
treponema  pallidum  and  that  the  antigen  for  the  Wassermann  test  would 
necessarily  consist  of  treponema  pallidum.  This  is  not  the  case.  Such  ambo- 
ceptors are  present,  in  varying  amounts  in  the  majority  of  syphilitic  sera.  They 
usually  constitute  not  more  than  10  or  20  per  cent,  of  the  syphilitic  amboceptors. 

The  amboceptors  almost  constantly  present  in  syphilitic  sera,  the  major 
amboceptor  content  of  such  sera,  are  specific  for  lipoid  or  lipoidal  substance. 
Consequently  the  ideal  antigen  for  the  Wassermann  test  is  composed  of  trepo- 
nema pallidum  and  lipoid  or  lipoidal  extracts  combined.  The  next  best  antigen, 
and  in  most  cases  entirely  sufficient,  is  a  lipoidal  extract  without  treponema. 
The  least  valuable  of  all  is  a  pure  culture  of  treponema. 

Combined  treponema  and  lipoid  extracts  are  obtained  by  extracting  the 
liver  or  liver  and  heart  of  a  syphilitic  fetus  with  alcohol.  Lipoid  antigens  are 
alcoholic  extracts  or  ether  soluble-acetone  insoluble  extracts  of  the  liver  or  liver 
and  heart  of  a  normal  fetus  or  a  normal  guinea-pig;  they  may  also  be  made  from 
ox  heart. 

When  the  method  of  preparation  is  identical,  different  extracts  vary  in  anti- 
genie  value,  whether  they  be  made  from  syphilitic  fetal  tissue,  normal  fetal 
tissue,  guinea-pig  tissue  or  ox  heart. 

The  antigenic  value  of  any  extract  can  only  be  determined  by  testing  it 
with  a  large  number  of  known  syphilitic  sera  and  non-syphilitic  sera,  hence  it  is 
necessary  in  the  beginning  to  procure  from  a  reliable  source  an  antigen  of  estab- 
lished value  and  standardized  strength.  The  new  antigens  prepared  by  the 
beginner  are  then  tested  by  repeated  comparison  with  the  original  and  when 
found  to  be  equal  to  the  original  are  stored  for  future  use. 

All  these  extracts  possess  two  properties:  antigenic  and  anticomplementary. 
The  first  is  desirable,  the  second  undesirable. 

In  this  connection,  we  may  define  antigenic  as  the  power  of  causing  fixation 
of  complement  in  the  presence  of  syphilitic  serum  and  in  no  other  case;  hence, 
will  show  no  hemolysis  with  a  syphilitic  serum  at  the  end  of  a  Wassermann  test, 
and  show  complete  hemolysis  at  the  end  of  a  Wassermann  test  with  all  other 
sera. 

Anticomplementary  may  be  defined  as  the  power  of  vitiating  complement 
under  any  and  all  circumstances,  when  used  in  sufficient  amount;  hence  showing 
no  hemolysis  at  the  end  of  a  Wassermann  test  both  with  syphilitic  and  non- 
syphilitic  sera. 

When  the  smallest  quantity  of  an  extract  that  is  anticomplementary  is  less 


244  MEDICAL  BACTERIOLOGY 

than  four  times  the  mean  antigenic  quantity  it  must  be  rejected  as  unfit  for 
use. 

Titration  shows  that  different  quantities  of  an  extract  are  antigenic,  e.g., 
from  o.oi  to  i.o  cc.  or  from  o.i  to  0.5  cc.,  that  is,  in  the  first  case,  any  quantity 
of  the  extract  from  o.oi  to  i.o  cc.  would  cause  complement  fixation  with  a  syphi- 
litic serum  and  have  no  effect  on  the  complement  with  non-syphilitic  sera;  in 
the  second  case  only  quantities  of  extract  from  o.i  to  0.5  cc.  would  do  this. 
In  the  first  case  the  mean  antigenic  quantity  would  be  0.5  cc.;  in  the  second  case 
the  mean  antigenic  quantity  would  be  0.3  cc. 

The  unit  of  antigen,  the  amount  used  in  making  Wassermann  tests,  is  the 
mean  antigenic  quantity.  It  is  determined  for  each  new  antigen  before  use, 
and  when  in  use  antigen  should  be  restandardized  at  least  every  month.  The 
method  of  standardization  will  be  described  later. 

CONTROLS 

The  end  result  and  accuracy  of  any  complement  fixation  test  depends  upon 
the  purity  and  potency  of  the  different  substances  used,  their  proper  combina- 
tion and  quantities,  the  time  and  temperature  of  incubation.  With  so  many 
sources  of  possible  error  it  is  imperative  that  each  be  controlled.  Nine  control 
tubes  should  be  carried  through  with  every  Wassermann  test  and  these  tubes 
should  always  have  the  same  positions  in  the  test-tube  rack.  The  author 
places  them  as  illustrated  in  Fig.  i .  Three  of  these  tubes  are  used  for  making  a 
Wassermann  test,  using  a  human  serum  that  was  obtained  from  a  patient,  who, 
so  far  as  medical  skill  could  determine,  never  had  syphilis,  a  serum  that  pre- 
viously showed  a  negative  Wassermann  reaction,  a  serum  that  does  not  in  any 
way  act  on  guinea-pig  serum,  rabbit  serum,  syphilitic  antigen  or  red  cells. 

Three  other  controls  are  used  for  making  a  Wassermann  test,  using  human 
serum  that  was  obtained  from  a  patient  that  presented  typical  positive  clinical 
signs  of  syphilis,  a  serum  that  previously  showed  a  positive  Wassermann  reac- 
tion, a  serum  that  does  not  in  any  way  act  on  guinea-pig  serum,  rabbit  serum, 
syphilitic  antigen  or  red  cells,  except  that  it  fixes  the  complement  in  guinea-pig 
serum  together  with  syphilitic  antigen. 

When  a  known  positive  serum  gives  a  positive  reaction,  and  a  known  nega- 
tive serum  gives  a  negative  reaction,  with  the  same  reagents,  in  the  same  quan- 
tities, and  under  the  same  conditions  as  the  unknown  sera  are  subjected  to,  it 
is  the  strongest  evidence  that  the  tests  have  been  properly  performed  and  the 
results  reliable — for  this  no  other  controls  are  necessary — but  sometimes  a  test 
goes  wrong,  and  these  controls  are  not  always  sufficient  to  indicate  the  source 
of  error;  for  this  purpose  three  other  controls  are  made:  one  for  complement,  one 
for  hemolytic  system  and  one  for  syphilitic  antigen  (Fig.  36). 

Known  positive  and  known  negative  sera,  procured  under  aseptic  conditions 
and  kept  sterile,  if  stored  in  a  refrigerator,  in  all  but  very  exceptional  cases  re- 
main fit  for  use  as  controls  for  several  weeks  or  months.  They  must  be  inacti- 
vated immediately  before  each  time  they  are  used;  if  this  is  not  done  they  are 
not  reliable. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  245 

For  each  serum  that  is  to  be  subjected  to  the  Wassermann  test  three  tubes 
are  used;  one  of  these  tubes  during  the  first  hour  of  incubation  contains  the  sus- 
pected patient's  serum  and  guinea-pig  serum  only,  no  syphilitic  antigen ;  this  is 
a  control  for  the  patient's  serum.  Only  sera  which  contain  nothing  that  can 
inhibit  hemolysis,  other  than  syphilitic  amboceptors,  are  susceptible  to  exami- 
nation by  the  complement  fixation  test.  Since  amboceptor  cannot  fix  comple- 
ment except  in  the  presence  of  antigen,  and  there  is  no  antigen  in  this  tube,  then 
the  complement  must  remain  free  and  unaltered,  regardless  of  whether  the  serum 
is  syphilitic  or  not. 

If  the  complement  in  this  tube  is  fixed  or  vitiated  during  the  first  hour  of 
incubation  it  is  evidence  that  the  human  serum  contains  something,  not  relative 
to  syphilis,  which  makes  impossible  its  examination. 

In  other  words,  the  tube  that  contains  no  syphilitic  antigen  must  always 
show  complete  hemolysis  at  the  end  of  a  Wassermann  test;  if  it  does  not  there 
is  no  diagnostic  significance  to  the  findings. 

The  writer  has  never  seen  a  human  serum  cause  this  phenomenon  when 
subjected  to  examination  within  3  days  after  withdrawal  from  the  circulation, 
but  human  sera,  even  when  sterile,  occasionally  do  so  to  a  slight  degree  several 
weeks  after  they  have  been  obtained  from  a  patient;  putrid  sera  nearly  always 
vitiate  complement.  This  control  is  therefore  indispensable. 

TECHNIQUE  OF  THE  WASSERMANN  TEST 

1.  Bleed  patient  or  patients. 

2.  Compute  the  number  of  tubes  that  will  be  used  in  making  the  tests; 
allowing  o.i  cc.  of  guinea-pig  serum  for  each  tube,  compute  the  amount  required. 

3.  Obtain  sufficient  guinea-pig  blood  to  yield  10  per  cent,  more  than  com- 
puted amount  of  serum  required. 

4.  Collect  blood  in  citrate  solution  to  furnish  red  cells. 

5.  Wash  and  make  a  5  per  cent,  suspension  of  the  red  cells  in  normal  salt 
solution. 

6.  Make  a  10  per  cent,  solution  of  the  guinea-pig  serum. 

7.  Make  a  i  per  cent,  solution  of  sensitized  rabbit  serum. 

8.  Titrate  or  standardize  the  complement. 

9.  Separate  patient's  serum  from  clot  and  inactivate  it. 

10.  Compute  the  amount  of  antigen  required  and  mix  the  alcoholic  extract 
with  normal  salt  solution. 

11.  Place  three  tubes  in  the  rack  for  each  patient's  serum  to  be  tested,  also 
three  for  the  positive  control  serum  and  three  for  the  negative  control  serum, 
place  three  tubes  at  the  extreme  right  for  complement  control,  hemolytic  sys- 
tem control,  and  antigen  control  (see  Fig.  36). 

12.  Put  one  unit  of  antigen  in  every  tube  in  the  middle  and  bottom  rows 
except  the  hemolytic  system  control  tube  and  the  antigen  control  tube,  put  no 
antigen  in  the  hemolytic  system  control  tube,  put  two  units  of  antigen  in  the 
antigen  control  tube,  do  not  put  antigen  in  any  tube  in  the  top  row. 

13.  Put  o.i  cc.  of  patient's  serum  in  each  of  the  three  tubes  provided  for  it, 


246  MEDICAL  BACTERIOLOGY 

put  o.i  cc.  of  positive  serum  in  each  of  the  three  tubes  provided  for  it,  put  o.i 
cc.  of  negative  serum  in  each  of  the  three  tubes  provided  for  it. 

14.  Put  one  unit  of  complement  in  all  the  tubes  in  the  top  and  middle  rows 
and  in  the  antigen  control  tube,  put  two  units  of  complement  in  all  the  tubes  in 
the  bottom  row,  except  the  antigen  control. 

15.  Shake  each  tube  to  thoroughly  mix  its  contents  and  place  in  incubator  at 
37°C.  for  i  hour. 

1 6.  Put  two  units  of  amboceptor  (rabbit  serum)  in  every  tube  except  the 
complement  control. 

17.  Put  i  cc.  of  the  5  per  cent,  suspension  of  red  cells  in  every  tube. 

1 8.  Shake  each  tube  to  thoroughly  mix  the  contents  and  replace  in  incubator 
for  i  hour. 

19.  Inspect  each  tube  and  record  appearance,  place  in  refrigerator  or  allow 
to  stand  at  room  temperature  for  several  hours  and  then  make  final  inspection 
and  record  reactions. 

INSPECTION  OF  THE  COMPLETED  WASSERMANN  TEST 

When  the  tubes  are  removed  from  the  incubator  at  the  end  of  the  second 
hour  of  incubation  the  complement  control  tube  should  be  inspected  first.  If 
it  shows  hemolysis  the  guinea-pig  serum  is  at  fault,  the  results  are  worthless  and 
the  test  must  be  repeated  with  new  guinea-pig  serum.  If  it  shows  no  hemolysis 
the  hemolytic  system  control  tube  is  next  inspected;  this  should  show  complete 
hemolysis;  if  it  does  not  an  insufficient  amount  of  complement  or  amboceptor 
has  been  used,  the  results  are  therefore  worthless  and  the  test  must  be  repeated. 
The  complement  and  hemolytic  system  controls  presenting  the  proper  appear- 
ance, inspection  of  the  antigen  control  follows.  Hemolysis  should  be  complete; 
if  it  is  not,  an  excessive  quantity  of  antigen  has  been  employed,  the  results  are 
worthless,  and  the  test  must  be  repeated  after  restandardization  of  the  antigen 
or  the  introduction  of  a  new  standardized  antigen. 

When  complement  control,  hemolytic  system  control,  and  antigen  control 
tubes  all  present  the  proper  appearance,  the  known  negative  serum  and  known 
positive  serum  controls  are  inspected.  The  tube  for  each  of  these,  containing 
no  antigen,  should  show  complete  hemolysis.  The  other  known  negative  tubes 
should  both  show  complete  hemolysis.  The  known  positive  tube  containing 
one  unit  of  complement  should  show  no  hemolysis,  the  other  may  show  com- 
plete, incomplete  or  no  hemolysis. 

Only  when  all  these  controls  are  correct  can  one  be  confident  that  technical 
errors  are  absent  (see  Fig.  36). 

Of  the  tubes  containing  the  serum  or  sera  under  examination,  those  without 
syphilitic  antigen  are  inspected  first,  they  must  show  complete  hemolysis, 
otherwise  they  are  not  susceptible  to  the  Wassermann  test. 

Next  in  order  is  inspection  of  the  tubes  containing  one  unit  of  complement. 
If  these  tubes  show  complete  hemolysis  it  is  evidence  of  a  negative  reaction;  in 
this  case  the  tubes  containing  two  units  of  complement  will  also  show  complete 
hemolysis. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  247 

When  the  tubes  containing  one  unit  of  complement  show  no  hemolysis  the 
reaction  is  positive,  indicative  of  syphilis;  in  this  case  the  tubes  containing  two 
units  of  complement  may  show  complete,  incomplete  or  no  hemolysis,  dependent 
upon  whether  the  o.i  cc.  of  syphilitic  serum  contained  just  enough  amboceptors 
to  fix  one  unit  of  complement  or  more  than  that  amount. 

Occasionally,  at  the  end  of  the  second  hour  of  incubation,  half  or  more  of  the 
red  cells  will  remain  unhemolized  in  the  tube  containing  one  unit  of  complement. 
If  incubated  longer  or  allowed  to  stand  at  room  temperature  for  6  or  8  hours 
hemolysis  may  eventually  be  complete  or  it  may  remain  incomplete.  A 
serum  which  gives  this  atypical  reaction  may  be  syphilitic  or  it  may  not.  Such 
a  reaction  must  be  reported  doubtful. 

PREPARATION  OF  ANTIGEN 

Originally  antigen  for  the  Wassermann  test  was  made  by  placing  a  finely 
hashed  liver  of  a  syphilitic  fetus  in  a  dark  bottle  with  five  times  its  volume 
of  normal  salt  solution  and  shaking  continuously  for  48  hours.  The  contents 
of  the  bottle  were  then  filtered  through  fine  gauze  and  0.25  per  cent,  of  tricresol 
added.  Such  extracts  are  not  as  commonly  employed  at  present  because  of  the 
discovery  that  alcoholic  extracts  can  be  more  easily  made,  are  more  stable  and 
in  the  majority  of  cases  are  equally  as  good.  Salt  solution  extracts  frequently 
deteriorate  so  as  to  be  useless  in  i  or  2  months,  some  remain  stable  for  years. 
It  would  seem  from  the  known  facts  in  regard  to  bacterial  antigens  in  general 
that  the  salt  solution  extract  of  syphilitic  tissue  is  the  only  extract  that  contains 
treponema  pallidum  antigen.  Be  that  as  it  may,  large  series  of  comparative 
tests  show  that  its  only  superiority  is  in  some  of  the  cases  giving  a  doubtful 
reaction  with  other  extracts. 

Alcoholic  extracts  are  the  most  commonly  employed  at  present  and  are  most 
easily  made.  The  method  of  preparation  is  the  same,  whether  syphilitic  fetal 
liver,  fetal  heart,  guinea-pig  liver  and  heart  or  ox  heart  is  used.  The  tissue  is 
passed  through  a  meat  grinder  or  cut  into  as  small  pieces  as  possible  with  scis- 
sors, fibrous  tissue  removed  and  discarded,  the  rest  ground  to  a  pulp  in  a  mortar 
and  put  in  a  ground-glass-stoppered  bottle;  10  times  its  volume  of  absolute 
alcohol  is  added,  the  bottle  hermetically  sealed  and  shaken  for  15  minutes.  It  is 
then  placed  in  an  incubator  at  37°C.  for  Todays,  being  shaken  for  5  or  10  minutes 
every  day.  At  the  end  of  this  time  it  is  filtered  through  paper  until  perfectly 
clear  and  free  of  suspended  particles  and  stored  in  a  dark  closet.  Immediately 
before  use  it  is  diluted  with  salt  solution,  but  as  the  salt  solution  dilution  tends 
to  deteriorate  in  several  days  or  weeks,  it  is  not  good  practice  to  dilute  the  alco- 
holic extract  except  as  required. 

Ether  soluble  acetone  insoluble  extracts,  usually  made  from  ox  heart,  have 
been  very  much  lauded  by  some  but  in  practice  show  little  if  any  superiority 
over  alcoholic  extracts.  The  technique  for  preparing  this  antigen  and  its  sup- 
posed superiority  has  been  fully  discussed  by  Noguchi  (Proc.  Soc.  Exper.  Med. 
and  Biol.,  1909,  vii,  55). 

Acetone  soluble  antigen  does  have  some  slight  superiority  over  the  others 


248  MEDICAL  BACTERIOLOGY 

in  that  it  tends  to  reduce  to  the  minimum  the  occurrence  of  hemolytic  and  anti- 
complementary  properties  of  the  antigen.  It  is  prepared  by  drying  the  ground- 
up  tissue,  in  vacuo,  over  sulphuric  acid,  afterward  placing  it  in  acetone,  20  cc. 
for  each  gram  of  tissue.  This  is  shaken  for  5  minutes  and  incubated  at  room 
temperature,  in  the  dark,  for  10  days.  It  is  then  filtered  through  paper  and  the 
nitrate  evaporated  at  37°C.  To  each  gram  of  the  residue  100  cc.  of  methyl 
alcohol  is  added  and  it  is  stored  at  room  temperature,  protected  from  light. 
Immediately  before  use  it  is  mixed  with  10  times  its  volume  of  normal  salt 
solution. 

Any  of  the  above  antigens  can  be  fortified  by  the  addition  of  0.25  Gm.  of 
cholesterin  to  100  cc.;  this  procedure  seems  undesirable  as  it  is  adding  another 
reagent,  unnecessarily,  to  a  test  already  too  complex. 

Schurmann,  Sachs,  Rondin  and  others  have  employed  various  chemicals 
and  colloids  as  antigen,  but  as  yet  no  chemical  antigen  has  been  found  to  equal 
the  tissue  extracts.  Varney  and  Baeslack  have  recommended  an  acetone  extract 
of  gummata  removed  from  rabbit  testicles  following  the  successful  inoculation  of 
the  rabbit  testicles  with  treponema  pallidum. 

STANDARDIZATION  OF  ANTIGEN 

For  the  standardization  of  antigen  one  requires  at  least  2  cc.  of  a  known 
positive,  syphilitic  serum  and  a  like  amount  of  negative,  non-syphilitic  serum. 
Two  rows  of  tubes  are  placed  in  a  test-tube  rack. 

A  i:  10  dilution  of  the  alcoholic  extract  to  be  standardized  is  made  by  mix- 
ing 2  cc.  of  it  with  1 8  cc.  of  normal  salt  solution. 

This  is  put  in  the  tubes  as  follows: 

o.i  cc.  in  first  tube  in  top  row  and  first  tube  in  bottom  row. 

0.2  cc.  in  second  tube  in  top  row  and  second  tube  in  bottom  row. 

0.3  cc.  in  third  tube  in  top  row  and  third  tube  in  bottom  row. 

0.4  cc.  in  fourth  tube  in  top  row  and  fourth  tube  in  bottom  row. 

0.5  cc.  in  fifth  tube  in  top  row  and  fifth  tube  in  bottom  row. 

0.6  cc.  in  sixth  tube  in  top  row  and  sixth  tube  in  bottom  row. 

0.7  cc.  in  seventh  tube  in  top  row  and  seventh  tube  in  bottom  row. 

0.8  cc.  in  eighth  tube  in  top  row  and  eighth  tube  in  bottom  row. 

0.9  cc.  in  ninth  tube  in  top  row  and  ninth  tube  in  bottom  row. 

1.0  cc.  in  tenth  tube  in  top  row  and  tenth  tube  in  bottom  row. 

1.1  cc.  in  eleventh  tube  in  top  row  and  eleventh  tube  in  bottom  row. 

1.2  cc.  in  twelfth  tube  in  top  row  and  twelfth  tube  in  bottom  row. 

Next  put  o.i  cc.  of  inactivated  syphilitic  serum  in  each  tube  at  the  bottom 
row;  put  o.i  cc.  of  inactivated  non-syphilitic  serum  in  each  tube  of  the  top  row. 

Put  one  unit  of  complement  in  every  tube  (both  rows).  Shake  each  tube  to 
thoroughly  mix  its  contents  and  place  in  incubator. 

When  the  tubes  have  been  incubated  exactly  i  hour,  add  one  unit  of  ambo- 
ceptor  (rabbit  serum)  and  i  cc.  of  red  cells  to  every  tube,  shake  to  mix  contents 
and  again  incubate  for  i  hour. 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  249 

Should  inspection  reveal  complete  hemolysis  in  all  the  tubes  in  the  top  row, 
and  no  hemolysis  in  all  the  tubes  in  the  bottom  row,  each  of  the  various  amounts 
of  extract  used  are  antigenic  and  the  extract  is  apparently  a  good  one.  But 
these  were  not  a  sufficient  number  of  tubes  to  show  the  smallest  quantity  that 
is  antigenic  nor  the  smallest  quantity  that  is  anticomplementary,  hence  the  test 
must  be  extended. 

Make  a  1:100  dilution  by  mixing  0.2  cc.  of  the  alcoholic  extract  with  19.8 
cc.  of  normal  salt  solution. 

Repeat  the  test  exactly  as  before,  except  that  the  1:100  dilution  is  used 
instead  of  the  1:10;  this  in  practically  all  cases  will  show  the  smallest  amount 
that  is  antigenic.  Amounts  of  extract  less  than  the  minimum  antigenic  quan- 
tity will  show  complete  hemolysis  in  the  tubes  containing  non-syphilitic  serum 
and  will  also  show  more  or  less  hemolysis  in  tubes  containing  syphilitic  serum. 

The  smallest  anticomplementary  quantity  is  yet  to  be  determined;  for  this 
purpose  repeat  the  test,  using  a  i :  5  dilution  of  the  alcoholic  extract  in  quantities 
from  0.5  cc.  in  the  first  tubes  to  1.6  in  the  last  tube.  Quantities  that  are  anti- 
complementary  show  no  hemolysis  in  the  tubes  containing  syphilitic  serum  and 
incomplete,  partial  or  no  hemolysis  in  tubes  containing  non-syphilitic  serum. 
The  quantity  of  antigen  to  be  used  in  making  Wassermann  tests — the  unit  of 
antigen — has  been  discussed  on  page  244. 

When  standardizing  antigen,  as  a  control,  Wassermann  tests  are  made  on 
the  human  sera  used  in  the  standardization,  and  these  tests  are  carried  out  with 
the  antigen  previously  in  use. 

SPINAL  FLUID 

In  certain  cases  it  is  desirable  to  subject  the  spinal  fluid  to  the  Wassermann 
test.  The  fluid  is  obtained  when  the  patient  is  in  bed,  lying  on  his  side  with  the 
body  bent  forward  and  thighs  flexed.  Under  strict  aseptic  precautions  (after 
local  anesthesia  if  desired)  a  strong  needle,  at  least  6  inches  long,  is  thrust  be- 
tween the  third  and  fourth  lumbar  vertebrae  into  the  spinal  canal.  As  the 
fluid  comes  out,  drop  by  drop,  it  is  caught  in  a  test-tube  until  5  cc.  have  been  col- 
lected; the  needle  is  then  withdrawn,  the  puncture  painted  with  tincture  of 
iodine  and  covered  with  a  sterile  dressing.  It  is  best  for  the  patient  to  remain 
at  rest  in  bed  for  at  least  several  hours.  In  the  absence  of  diseases  other  than 
syphilis  the  gross  appearance  of  the  spinal  fluid  is  practically  normal — clear, 
almost  colorless  and  like  water.  Should  it  show  any  turbidity  it  is  well,  if  not 
necessary,  to  centrifugalize  it.  Spinal  fluid,  like  blood  serum,  should  be  pro- 
tected from  contamination,  examined  if  possible  within  48  hours  after  its  removal 
from  the  patient,  and  inactivated  by  heating  in  a  water  bath  at  55°C.  for  J^ 
hour  before  testing. 

The  technique  is  the  same  when  making  a  Wassermann  test  with  spinal 
fluid  as  with  blood  serum,  except  that  six  tubes  are  used  each  of  which  contains 
one  unit  of  complement  and  the  first  two  tubes  each  contain  o.i  cc.  of  spinal 
fluid,  the  third  tube  0.2  cc.  of  spinal  fluid,  the  fourth  tube  0.3  cc.  of  spinal  fluid, 


250  MEDICAL  BACTERIOLOGY 

the  fifth  tube  0.4  cc.  of  spinal  fluid,  and  the  sixth  tube  0.5  cc.  of  spinal  fluid. 
The  reason  for  this  variation  will  be  discussed  in  the  following  pages. 

THE  SIGNIFICANCE  OF  THE  WASSERMANN  REACTION 

Probably  no  test  has  been  subjected  to  a  greater  amount  of  investigation 
to  detect  and  correct  its  faults  and  determine  its  value  and  proportionate  weight 
in  diagnosis.  While  there  is  much  about  it  not  understood  at  present  and  per- 
haps more  to  be  learned,  certain  facts  have  been  clearly  established,  which  show 
this  test  to  be  of  inestimable  value. 

There  are  factors,  some  of  which  are  recognizable  and  removable,  which  at 
times  cause  the  blood  serum  or  spinal  fluid  of  a  patient  infected  with  syphilis 
to  give  a  negative  Wassermann  reaction.  First  among  these  to  be  considered 
is  the  stage  of  the  disease: 

In  primary  syphilis  (from  the  appearance  of  the  chancre  until  the  appear- 
ance of  the  rash)  the  serum  of  most  syphilitic  patients  gives  a  negative  reaction 
in  the  first  week,  many  give  a  negative  in  the  second  week,  some  give  a  negative 
result  in  the  third,  fourth  and  fifth  weeks;  nearly  all  give  a  positive  reaction 
after  this  time. 

In  early  secondary  syphilis,  when  the  disease  is  active,  nearly  100  per  cent, 
give  a  positive  reaction;  when  the  disease  is  latent  from  5  to  20  per  cent,  give  a 
negative  reaction. 

In  tertiary  syphilis,  when  active,  from  i  to  10  per  cent,  give  a  negative  reac- 
tion; when  latent,  from  20  to  25  per  cent,  give  a  negative  reaction;  when  the 
reaction  is  negative  in  latent  syphilis  provocative  treatment  will  frequently 
produce  a  positive  reaction. 

When  the  blood  serum  gives  a  negative  reaction  in  late  secondary  and 
tertiary  syphilis,  the  spinal  fluid  frequently  gives  a  positive  reaction. 

In  a  small  per  cent.,  probably  less  than  2,  of  paresis  both  the  blood  serum  and 
spinal  fluid  give  a  negative  reaction. 

The  serum  of  a  syphilitic  patient,  obtained  during  or  shortly  after  the  patient 
has  been  profoundly  intoxicated  with  alcohol,  will  frequently  give  a  negative 
reaction. 

When  a  syphilitic  patient  has  received  sufficient  mercury  or  salvarsan  to 
partially  or  completely  subdue  signs  and  symptoms  of  pathological  activity, 
even  though  the  disease  is  destined  to  later  manifest  its  presence,  the  reaction 
will  frequently  be  negative  for  3  weeks  after  the  treatment  has  been  stopped, 
occasionally  negative  for  6  weeks  and  rarely  negative  for  longer  periods,  up  to  a 
year. 

It  is  possible  that  some  infections,  secondary  to  syphilis,  may  cause  a  tem- 
porary or  permanent  suppression  of  a  positive  reaction.  This  has  not  been  dis- 
covered but  is  mentioned  as  a  possibility  to  be  considered  in  certain  cases  and 
a  lead  to  desirable  research. 

It  is  impossible  to  determine  yet  in  what  per  cent,  of  dormant  congenital 
syphilis  the  reaction  is  negative;  the  results  so  far  obtained  strongly  indicate 
that  it  is  small. 


WASSERMANN  AND    OTHER   COMPLEMENT   FIXATION   TESTS  251 

Nearly  all  cases  of  active,  secondary  and  congenital  syphilis  give  strong 
positive  reactions  when  not  influenced  by  medication  or  alcoholic  intoxication. 
In  latent  or  dormant  cases  of  secondary  and  congenital  syphilis,  strong  positive 
reactions  are  common,  but  taken  as  a  whole  they  are  less  pronounced  than  in  the 
active  secondary  stage;  and  in  latent  tertiary  syphilis,  the  greatest  number  of 
weak  positive  reactions  are  observed;  frequently  the  reactions  are  so  slight  that 
they  must  be  considered  doubtful. 

As  to  the  significance  of  a  clear-cut  positive  reaction  little  need  be  said,  yaws 
and  leprosy  are  the  only  diseases  other  than  syphilis  that  can  cause  a  positive 
reaction;  having  excluded  these,  and  technical  error,  a  positive  Wassermann 
practically  always  indicates  syphilis. 

The  amount  of  amboceptor  present  in  the  serum  of  a  syphilitic  patient 
gradually  increases  from  zero  in  the  period  of  incubation,  to  many  times  the 
amount  necessary  to  fix  all  the  complement  in  o.i  cc.  of  guinea-pig  serum  (using 
o.i  cc.  of  the  patient's  serum)  in  the  secondary  stage  of  the  disease.  It  tends  to 
gradually  decrease  in  the  tertiary  stage  and  may  fluctuate  as  the  disease  is 
influenced  by  treatment. 

As  a  consequence  all  degrees  of  reaction  are  observed  in  Wassermann  tests, 
made  with  syphilitic  sera,  from  no  complement  fixation  to  complete  complement 
fixation. 

With  our  present  knowledge  of  the  serology  of  syphilis  there  is  little  chance 
of  error  in  interpreting  the  significance  of  a  Wassermann  reaction  when  all  or 
none  of  the  complement  is  fixed,  but  when  very  slight  amounts  of  complement 
are  fixed,  when  hemolysis  is  partial,  not  complete;  in  other  words,  when  the 
reaction  is  doubtful,  misinterpretations  are  inevitable.  Fortunately,  the  patients 
giving  such  reactions  are  few  in  comparison  to  the  whole;  but  they  embrace  a 
considerable  number  of  syphilitics.  Erroneous  conclusions  in  these  cases  can 
be  minimized  by  repeated  examinations  of  the  patient's  serum  at  weekly  or 
fortnightly  intervals  and  making"  the  tests  with  several  different  antigens  and 
proportioning  the  reagents  so  as  to  make  the  test  most  sensitive — but  error  can- 
not be  entirely  precluded;  herein  lies  the  greatest  weakness  of  the  test. 

Some  apparently  normal  individuals  and  some  non-syphilitic  patients,  espe- 
cially those  afflicted  with  any  of  the  diseases  classified  as  "  chronic  granulomata" 
have  in  their  blood  serum  something  which  tends  to  deviate,  vitiate  or  fix 
complement. 

This  non-specific  deviation  of  complement  can  usually  but  not  always  be 
measured  by  placing  o.i  cc.  of  the  suspected  serum  (inactivated)  in  each  of  a 
number  of  tubes,  adding  one  unit  of  hemolytic  amboceptor  and  one  unit  of  red 
cells  to  each  tube,  and  beginning  with  one  unit  of  complement  in  the  first  tube 
add  gradually  increasing  amounts  of  complement  to  successive  tubes;  shake, 
incubate  for  i  hour  and  then  discover  the  smallest  amount  of  complement  which 
gives  complete  hemolysis;  the  Wassermann  test  is  then  made,  using  this  amount 
of  complement  as  one  unit. 

Occasionally  a  serum  that  gives  a  doubtful  reaction  with  a  salt  solution  ex- 
tract antigen  will  give  a  clear-cut  negative  or  positive  with  an  alcoholic  or  ether 


252  MEDICAL  BACTERIOLOGY 

soluble-acetone  insoluble  or  acetone  soluble  or  cholesterinized  antigen — or  the 
reverse,  so  in  doubtful  cases  repetition  of  the  test  with  these  different  antigens 
may  distinguish  its  true  nature. 

The  employment  of  two  units  of  hemolytic  amboceptor  is  necessary  in  rou- 
tine complement  fixation  to  avoid  error  from  non-specific  deviation  of  comple- 
ment. When  a  doubtful  reaction  is  obtained  if  the  test  is  repeated  using  only 
one  unit  of  hemolytic  amboceptor,  after  a  preliminary  examination  of  the  sus- 
pected serum  for  non-specific  complement  deviation  properties,  a  decided  posi- 
tive reaction  is  frequently  obtained. 

I  have  carefully  studied  this  phase  of  the  subject  and  believe  accuracy  is 
more  nearly  realized  and  the  interests  of  patients  best  served  by  adhering  to  the 
following  rule  when  the  Wassermann  reaction  is  doubtful : 

If  the  patient  has  previously  had  unquestionable  history,  physical  signs  or 
serum  reaction  of  syphilis,  consider  it  positive. 

If  the  patient's  history  contraindicated  syphilis  and  there  are  no  positive 
clinical  evidences  of  the  disease,  eliminate  the  possibility  of  the  reaction  having 
been  masked  by  mercury  or  arsenic  or  alcohol,  give  provocative  treatment,  ob- 
tain both  blood  and  spinal  fluid  and  examine  both,  using  salt  solution  fetal  liver 
extract,  alcoholic  extract  and  ether  soluble-acetone  insoluble  antigens  and  two 
units  of  hemolytic  amboceptor.  If  under  these  conditions  more  than  half  of  the 
cells  are  hemolized  in  the  tube  containing  o.i  cc.  of  patient's  serum  and  one  unit 
of  complement,  and  there  is  complete  hemolysis  in  the  tube  containing  two  units 
of  complement — reserve  diagnosis  and  institute  salvarsan  treatment — if  this 
does  npt  alter  the  reaction,  consider  it  negative. 

It  is  the  consensus  of  opinion  that  the  Wassermann  test  is  one  of  the  greatest 
factors  in  medical  diagnosis.  Sporadically,  prominent  men  attempt  to  belittle 
its  value.  He  who  will  trouble  to  investigate  will  soon  learn  that  the  great 
majority  of  congenital  syphilitics,  unrecognized  as  such  by  physical  examina- 
tion are  properly  diagnosticated  by  this  test;  that  in  cases  of  doubtful  nature, 
which  if  syphilitic  are  in  the  tertiary  stage,  the  diagnosis  without  the  Wasser- 
mann test  is  as  often  wrong  as  right,  and  with  it  the  error  is  reduced  one- 
half.  These  are  not  its  greatest  merits,  but  suffice  to  make  it  an  indispensable 
part  of  every  conscientious  physician's  armamentarium. 

FURTHER  CONSIDERATION  OF  THE  WASSERMANN  TEST 

In  the  development  of  the  complement  fixation  test  for  syphilis,  Wassermann 
and  his  co-workers  investigated  each  factor  entering  into  it  and  the  mechanism 
of  reaction  more  thoroughly  than  any  of  their  followers.  In  his  original  com- 
munication he  states  there  is  a  fundamental  reason  for  each  step  in  the  technique 
he  describes  and  this  has  been  amply  proven. 

Before  the  profession  at  large  knew  of  the  test  several  modifications  were 
introduced  by  as  many  different  experimenters.  So  many  modifications  have 
been  recommended  that  it  would  require  a  large  volume  to  tabulate  them.  The 
numerous  modifications  have  one  feature  in  common,  namely,  they  have  all 
failed  to  stand  the  test  of  time.  Each  has  had  its  enthusiastic  advocates  but 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  253 

comparison  with  the  original  Wassermann,  through  long  series  of  tests,  has  even- 
tually established  the  superiority  of  the  Wassermann  test. 

The  experience  of  years  of  universal  application  of  the  Wassermann  test 
has  naturally  amplified  knowledge  of  the  different  elements  entering  into  it  and 
established  certain  facts  that  have  permitted  slight  changes  in  technique,  in  no 
case  basic,  but  such  as  to  a  slight  degree  simplify  the  performance  of  the  test — • 
as  the  introduction  of  alcohol,  ether  and  acetone  extracts  of  tissue  for  antigen, 
the  reduction  of  the  quantities  used  in  the  test  to  one-half  the  original  volumes, 
the  reduction  of  incubation  from  2  hours  to  i,  and  the  introduction  of  quantita- 
tive determination  of  the  syphilitic  reaction. 

The  quantity  of  any  one  of  the  five  substances  entering  into  a  complement 
fixation  test  must  bear  a  proportionate  relation,  within  certain  limits,  to  all  the 
others.  Theoretically,  the  larger  the  quantity  of  patient's  serum  used  the 
greater  the  possibility  of  detecting  syphilitic  antibodies  when  present  in  small 
amount.  The  amount  of  human  serum  that  may  be  used  is  limited,  however, 
by  the  irregular  but  frequent  presence  in  human  serum  of  substances,  not  related 
to  syphilis,  which  tend  to  deviate  complement.  The  proportion  of  human 
serum  to  other  substances  in  the  test  as  laid  down  by  Wassermann  is  such  that 
this  non-specific  deviation  of  complement  cannot  cause  positive  reaction  in 
negative  cases.  Human  serum  also  may  contain  substances  which  can 
hemolize  red  cells,  without  the  presence  or  aid  of  complement;  in  the  propor- 
tion of  human  serum  to  red  cells  used  in  the  Wassermann  test  this  effect  is 
not  observed,  but  if  four  or  five  times  as  much  human  serum  is  used  it  is 
frequently  noted,  this  would  give  a  negative  reaction  (hemolysis)  even  though 
the  serum  was  positive,  therefore,  blood  serum  cannot  be  examined  as  spinal 
fluid  is.  When  serum  is  not  inactivated  before  subjecting  it  to  a  complement 
fixation  test,  a  negative  serum  frequently  shows  a  positive  reaction;  this  is 
especially  true  when  the  antigen  contains  protein. 

A  reduction  of  the  quantity  of  human  serum  used  to  make  a  Wassermann 
test  necessitates  a  proportionate  reduction  in  the  quantity  of  all  the  other  sub- 
stances; the  only  advantage  in  such  a  procedure  is  a  reduction  in  labor  and  ex- 
pense which  necessarily  is  associated  with  a  greater  probability  of  error,  because 
no  matter  how  careful  a  worker  may  be  the  smaller  the  quantities  with  which  he 
works  the  greater  the  proportion  of  error  in  measurements;  in  addition  to  this, 
the  Wassermann  test  seems  to  be  a  chemical  reaction  of  colloidal  nature  and 
this  further  accentuates  the  danger  of  error  in  greatly  reducing  the  quantities 
used  in  making  the  test. 

A  comparatively  small  amount  of  experimentation  will  convince  one  that 
human  serum  should  be  inactivated  and  that  not  less  than  o.  i  cc.  nor  more  than 
0.2  cc.  should  be  used. 

The  sensitiveness  of  the  Wassermann  test,  the  power  to  show  distinct  posi- 
tive reactions  with  human  serum  containing  a  very  small  quantity  of  specific 
amboceptor,  can  be  increased  by  using  amounts  of  antigen  but  slightly  less  than 
the  minimum  anticomplementary  quantity. 

The  advantage  so  gained  in  obtaining  positive  reactions  on  syphilitic  serum 


254  MEDICAL  BACTERIOLOGY 

which  otherwise  would  show  a  doubtful  or  negative  reaction  is  more  than  offset 
by  the  frequency  of  spurious  positive  reactions,  obtained  with  non-syphilitic 
sera  by  this  method.  This  is  the  worst  fault  any  test  can  have,  hence  it  is  un- 
justifiable to  make  the  test  more  sensitive  by  increasing  the  amount  of  antigen 
used  to  more  than  one-fourth  the  minimum  anticomplementary  quantity. 

Antigen,  in  whatever  quantity  employed,  to  some  degree  exerts  an  inhibiting 
effect  on  complement;  human  serum  does  likewise;  when  human  serum  and  anti- 
gen are  combined  the  total  inhibiting  effect  is  greater  than  the  sum  of  both 
acting  separately.  The  non-specific  anticomplementary  factor  varies  in  the 
sera  of  different  individuals.  Within  certain  limitations,  described  as  the  Neis- 
ser-Wechsberg  phase,  a  deficiency  of  complement  can  be  made  up  for  by  an 
excess  of  amboceptor;  therefore,  if  two  units  of  amboceptor  are  used  in  the 
Wassermann  test  there  is  always  sufficient  compensation  of  the  non-specific 
deviation  of  complement  by  human  serum  and  antigen  and  false  positive  reac- 
tions are  precluded.  The  disadvantage  of  this  method  of  compensation  is  that 
frequently  serum  under  examination  contains  not  quite  enough  syphilitic  ambo- 
ceptor to  fix  one  unit  of  complement  and  the  anticomplementary  factor  of  the 
human  serum  and  antigen  is  low,  so  that  the  excess  of  amboceptor  is  sufficient 
to  bring  about  a  doubtful  or  negative  reaction  although  the  serum  does  contain 
a  small  amount  of  syphilitic  amboceptor. 

The  temptation  to  increase  the  sensitiveness  of  the  test  by  using  one  unit  of 
amboceptor  is  too  strong  for  many,  but  it  is  a  baneful  practice  bound  to  elicit 
numerous  pseudopositive  reactions. 

It  is  possible  to  measure  the  combined  human  serum  and  antigen  non-specific 
deviation  of  complement;  to  do  so  is  a  procedure  that  consumes  an  amount  of 
time  and  labor  that  forbids  it  as  a  routine  procedure.  In  the  comparatively 
few  cases  where  the  use  of  two  units  of  amboceptor  give  unsatisfactory  results 
and  it  is  desirable  to  perform  the  test  using  one  unit,  the  non-specific  deviation 
of  complement  should  first  be  measured  and  compensated  for  by  the  addition  of 
the  required  amount  of  complement. 

The  selection  of  rabbit  serum  containing  hemolytic  amboceptor  is  worthy 
of  note.  In  a  previous  chapter  it  has  been  stated  that  no  serum  should  be  used 
if  more  than  o.oi  cc.  was  required  to  furnish  a  unit  of  amboceptor.  It  is  not 
difficult  to  produce  a  serum  10  or  20  times  as  strong,  so  that  o.ooi  cc.  contains 
a  unit  of  amboceptor.  The  greater  the  amboceptor  content  the  more  desirable 
is  the  serum  because,  when  a  rabbit  is  injected  with  sheep  cells,  precipitins  and 
agglutinins  as  well  as  lysins  are  formed;  when  the  lysin  (amboceptor)  content  is 
low  the  agglutinin  and  precipitin  content  is  proportionately  very  much  greater 
than  when  the  lysin  content  is  high;  therefore,  if  a  serum  poor  in  lysins  is  em- 
ployed precipitation  and  agglutination  of  the  red  cells  occur  and  to  some  degree 
inhibit  hemolysis.  Furthermore,  rabbit  serum,  like  all  other  sera,  may  nor- 
mally contain  substances  which  tend  to  inhibit  hemolysis  and  the  possibility  of 
such  an  occurrence  is  precluded  when  very  small  quantities  are  employed. 

Generally  the  quantity  of  antibodies  per  cubic  centimeter  in  serum  is  in  pro- 
portion to  the  severity  of  an  infection  or  the  mass  of  inoculation;  the  greater  the 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  255 

quantity  of  antigen  inoculated,  the  greater  the  number  and  virulence  of  infective 
organisms,  the  greater  the  antibody  production.  Exceptions  to  this  rule  are 
numerous,  especially  and  conspicuously  so  in  syphilis.  Any  complement  fixation 
test  can  be  made  quantitative  as  well  as  qualitative.  Quantitative  Wasser- 
mann  tests  are  commonly  made,  usually  four  or  five  different  degrees  being  dis- 
tinguished; twenty  or  thirty  different  degrees  instead  of  five  might  quite  as  well 
be  recognized.  The  quantitative  determination  is  made  by  adding  different 
amounts  of  the  patient's  serum  to  a  fixed  amount  of  complement  or  different 
amounts  of  complement  to  a  fixed  amount  of  serum. 

The  majority  of  syphilitic  individuals  have  sufficient  amboceptors  in  o.i  cc. 
of  their  serum  to  fix  all  the  complement  in  the  same  amount  of  guinea-pig  serum; 
many  have  more  and  some  less.  Citron  recognized  this  fact  early  in  the  history 
of  the  test.  The  possibility  of  making  the  Wassermann  test  more  sensitive  by 
modifying  the  original  technique  and  that  given  in  previous  chapters  has  been 
described — also  the  accompanying  danger  of  obtaining  pseudopositive  reac- 
tions. If  one  believes  it  best  to  so  conduct  the  test  that  a  positive  reaction 
will  never  be  obtained  with  a  non-syphilitic  patient's  serum,  the  quantitative 
determination  is  limited  to  distinguishing  between  sera  containing  slightly 
less  than  the  average  amount  of  specific  amboceptors  and  those  containing  two, 
three  or  four  times  as  much,  or  more. 

It  must  be  obvious  to  anyone  familiar  with  the  nature  of  the  Wassermann 
test  that  an  accurate  quantitative  reaction  can  only  be  obtained  after  a  prelimi- 
nary determination  of  non-specific  complement  deviation  and  compensation  for 
same;  this  is  practically  never  done  in  routine  work — it  cannot  be — and  therefore 
routine  quantitative  determinations  are  nothing  more  than  rough  estimates, 
indicating  that  the  reaction  is  strong  or  weak  as  compared  to  the  average  reac- 
tion. It  is  desirable  to  recognize  whether  a  reaction  is  weak  or  strong  before 
treatment  is  instituted  so  that  future  tests  will  show  whether  the  treatment 
has  been  effective  or  not,  but  this  is  of  little  real  value  because  experience  has 
shown  that  no  treatment  is  permanently  effective  that  does  not  cause  a  complete 
disappearance  of  specific  amboceptors  and  hence  a  clear-cut  negative  reaction. 
I  believe  the  practice  of  reporting  reactions  as  +  ,  ++,  +  +  +  ,  +  +  +  +  ,  is- 
bad;  it  is  implying  a  difference  which  is  demonstrable  serologically  but  not  other- 
wise and  it  has  led  to  a  very  general  and  totally  erroneous  opinion  among  phy- 
sicians that  there  is  a  different  pathological,  therapeutic  and  prognostic  signifi- 
cance according  to  the  strength  or  weakness  of  the  reaction.  The  Wassermann 
test,  clinical  and  postmortem  findings  are  in  perfect  harmony  in  showing  that 
syphilis  requires  the  most  intensive  treatment  possible,  medication  to  the 
patient's  limit  of  toleration,  whether  the  reaction  be  +  or  +  +  +  +  +  ;  that  it  is 
frequently  just  as  difficult,  sometimes  more  difficult,  to  change  a  -j-  to  — ,  as  to 
change  a  +  +  +  +  +  to  — ;  that  among  the  virulent  cases  that  fail  to  respond 
to  treatment  and  terminate  fatally,  many  show  a  -f-  or  +  +  throughout  the 
disease  and  not  a  +  +  +  +  . 

There,  has  been  occasional  mention  in  the  literature  of  "  Wassermann-fast " 
cases,  patients  having  contracted  syphilis  and  developed  syphilitic  antibodies 


256  MEDICAL  BACTERIOLOGY 

which  persisted  in  the  blood  undiminished  by  treatment  and  for  long  periods 
after  cessation  of  treatment,  after  the  disappearance  of  all  signs  and  symptoms 
of  the  disease,  persons  apparently  in  good  health  and  looked  upon  as  having 
recovered.  I  have  never  discovered  such  a  case;  if  they  do  occur  they  are  very 
few.  Every  patient  I  have  seen  who.  persistently  gave  a  positive  Wassermann 
in  spite  of  active  treatment  has  had  active  syphilis,  usually  of  the  central 
nervous  system,  and  with  one  exception  all  have  died  within  3  years  after  com- 
ing under  observation. 

COMPLEMENT  FIXATION  TESTS  IN  THE  DIAGNOSIS  OF  INFECTIOUS  DISEASES 

OTHER  THAN  SYPHILIS 

In  most,  if  not  all,  infectious  diseases  amboceptors  capable  of  fixing  comple- 
ment in  the  presence  of  the  offending  organism  are  present  in  the  blood  serum. 
In  most  diseases  other  than  syphilis  the  amboceptors  are  not  so  regularly  or  con- 
stantly present  in  sufficient  amount  to  be  demonstrable  by  a  technique  less  sen- 
sitive than  will  elicit  pseudopositive  reactions,  hence  complement  fixation  tests 
in  most  diseases  are  not  so  valuable  as  other  tests  in  establishing  the  nature  of 
the  infection.  Probably  future  discoveries  will  greatly  extend  the  field  of  use- 
fulness of  complement  fixation  tests. 

Complement  fixation  tests  may  also  be  employed  to  determine  the  identity 
of  an  unknown  antigen.  No  matter  for  what  purpose  this  method  of  investi- 
gation is  used,  the  principle  is  the  same,  the  inherent  non-specific  properties  of 
the  substances  entering  into  the  test  are  the  same,  the  danger  of  errors  and  the 
methods  of  precluding  them  are  the  same  and  consequently  the  technique  should 
be  the  same. 

Next  to  syphilis  the  complement  fixation  test  is  most  frequently  used  in  the 
diagnosis  of  gonococcus  infections;  not  the  acute  suppurative  infections  of  the 
eye,  urethra  or  vagina,  but  in  those  cases  where  the  heart,  the  Fallopian  tubes, 
ovaries,  testicles,  seminal  vesicles  or  the  articulations  are  involved  sometime 
after  the  subsidence  of  disease  at  the  atrium  of  infection.  In  'the  absence  of 
these  complications  or  sequelae  the  test  is  also  made  at  times  to  determine 
whether  or  not  the  body  is  free  of  gonococci  after  the  subsidence  of  gonorrhea 
and  the  disappearance  of  gonococci.  The  test  has  a  distinct  value,  a  positive 
reaction  obtained  3  months  after  apparent  recovery  or  during  the  course  of  an 
arthritis  or  endocarditis  practically  always  indicating  persistent  infection,  but 
it  does  not  have 'so  great  a  value  as  the  Wassermann  has  in  the  diagnosis  of 
syphilis,  because  a  much  larger  number  of  persons  infected  with  the  gonococcus 
give  a  negative  reaction. 

In  making  a  complement  fixation  test  for  gonococcus  infection  the  technique 
is  exactly  the  same  as  the  Wassermann  except,  of  course,  gonococcus  antigen 
not  syphilitic  antigen  is  used  and  the  gonococcus  antigen  must  be  titrated  im- 
mediately before  each  test. 

Antigen  for  gonococcus  complement  fixation  tests  is  made  by  culturing  the 
gonococcus  on  Wertheim's  or  other  appropriate  media  at  37°C.  for  i  or  2  days, 
washing  the  growth  off  with  normal  salt  solution,  shaking  the  salt  solution 


WASSERMANN   AND    OTHER   COMPLEMENT   FIXATION   TESTS  257 

suspension  of  gonococci  vigorously  for  15  minutes  or  longer  and  adding  0.25  per 
cent,  tricresol;  keep  in  full  dark  containers  in  ice  box. 

.The  amount  of  salt  solution  used  to  wash  off  the  growth  and  suspend  it  is 
such  that  the  contained  bacteria  make  it  slightly  cloudy  or  turbid.  The  method 
of  standardization  and  titration  is  the  same  as  previously  described  for  Wasser- 
mann  antigen  except  that  one-half  the  minimum  anticomplementary  quantity 
is  taken  for  the  unit,  the  amount  to  be  used  in  making  tests.  The  value  of  a 
gonococcus  antigen  depends  to  a  considerable  degree  upon  its  being  polyvalent 
—including  half  a  dozen  or  more  strains  of  gonococci;  this,  together  with  the 
difficulty  of  culturing  the  organism,  compels  most  serologists  to  depend  upon 
some  distributing  laboratory  for  their  supply.  I  have  found  the  gonococcus 
antigens  marked  by  Parke,  Davis  &  Co.,  and  H.  K.  Mulford  Co.  as  good  as 
any  and  superior  to  most. 

The  complement  fixation  test  is  a  valuable  aid  in  the  diagnosis  of  echino- 
coccus  infestations;  the  filtered  fluid  from  an  echinoccus  cyst,  to  which  0.25 
per  cent,  of  tricresol  has  been  added,  is  used  for  antigen. 

The  detection  of  glanders  and  epidemic  abortion  of  cattle  is  largely  done  by 
complement  fixation  tests. 

Early  discovery  of  active  tuberculosis  is  one  of  the  most  important  and 
difficult  tasks  of  the  physician.  All  available  methods  of  investigating  these 
cases  are  frequently  inadequate.  A  test  that  can  disclose  the  nature  of  the 
disease  a  short  time  after  it  becomes  active,  before  serious  permanent  injury 
has  been  effected — and  will  indicate  subsidence  of  the  disease — will  supply  one 
of  the  greatest  aids  for  further  study  and  control  of  tuberculosis.  The  evidence 
accumulated  during  the  last  5  years  seems  to  indicate  that  such  a  test  will  be 
developed  in  the  near  future,  and  that  it  will  be  a  complement  fixation  test.  It 
has  been  well  established  that  a  demonstrable  quantity  of  specific  amboceptor 
is  present  in  the  blood  serum  of  a  large  per  cent,  of  patients  in  the  early  stages 
of  pulmonary  tuberculosis,  a  satisfactory  antigen  for  routine  clinical  examina- 
tions has  not  yet  been  made,  when  this  is  done  complement  fixation  tests  in  the 
diagnosis  of  tuberculosis  will  take  a  place  as  important  as  the  Wassermann  holds. 

BIBLIOGRAPHY 

The  serum  reactions  described  in  this  brief  work  are  a  very  small  portion  of 
the  subject  of  immunity.  The  best  understanding  of  these  reactions  and  their 
significance  requires  some  knowledge  of  the  subject  in  general. 

Any  attempt  to  present  so  complicated  a  subject  to  students,  in  a  brief 
time,  so  they  can  master  technique  that  will  be  available  in  diagnosis,  must  be 
to  some  degree  dogmatic  and,  therefore,  lacking  the  broadest  discussions  and 
presentation  of  facts  desirable.  With  this  in  mind,  the  student  inspired  with 
true  scientific  thought  is  most  earnestly  solicited  to  read  other  books  and  mono- 
graphs relative  to  the  subject,  especially  those  tabulated: 

"Studies  in  Immunity,"  Ehrlich-Bolduan,  2d  edition,  John  Wiley  &  Sons,  New  York. 
Deutsch.  med  Wchnschr.,  1906,  xxxii,  745  (original  Wassermann  monograph). 
17 


258  MEDICAL  BACTERIOLOGY 

"The  Wassermann  Reaction  in  the  Pathology,  Diagnosis  and  Treatment  of  Syphilis,"  Pearce, 
Richard  M.,  Archives  Int.  Med.,  1910,  vol.  vi,  pp.  478-516. 

"The  Effect  of  Treatment  on  the  Wassermann  Reaction,"  Swift,  Homer  F.,  Archives  Int. 
Med.,  1910,  vol.  vi,  pp.  626-637. 

"The  Use  of  PureLipoids  and  Alcoholic  Extracts  with  Active  and  Inactive  Serum  in  the  Com- 
plement-Fixation Tests  for  Syphilis,"  MacRae,  Eisenbrey  and  Swift,  Archives  Int.  Med., 
1910,  vol.  vi,  pp.  469-477. 

"A  Comparative  Study  of  Antigens  for  the  Wassermann  Reaction,"  Vareny  and  Baeslack, 
Jour.  A.  M.  A.,  Sept.  6,  1913. 

"Serum  Diagnosis  of  Syphilis,"  H.  Noguchi,  J.  B.  Lippincott  Co.,  Philadelphia. 

"The  Titration  of  Wassermann  Reagents,"  Lloyd  Thompson,  Archives  Int.  Med.,  June  15, 

1914,  vol.  xxiii,  No.  6. 

"A  Modified  Wassermann,"  Thompson,  Archives  Int.  Med.,  1913,  vol.  xi,  p.  512. 

"The  Coagulation  Test  for  Syphilis,"  H.  P.  Cole  and  S.  Eng.-Kin  Chin,  Archives  Int.  Med., 

1915,  vol.  xvi,  No.  5,  880. 

Amer.  Jour,  of  the  Med.  Sciences,  1911,  vol.  141.  p.  693  (Schwartz  and  McNeil,  original  mono- 
graph on  "Complement  Fixation  Test  for  Gonorrhea"). 

"Gonococcus  Complement  Fixation:  A  New  Lipoid  Antigen,"  Warden,  C.  C.,  and  Schmidt, 
L.  E.,  Jour.  Laboratory  and  Clin.  Med.,  February,  1916,  vol.  i,  No.  5,  p.  333. 

''Complement  Fixation  in  Tuberculosis,"  Stimson,  A.  M.,  Surg.  U.  S.  Pub.  Health  Service, 
Hygienic  Laboratory  Bulletin,  No.  101,  U.  S.  Pub.  Health  Service,  Washington,  D.  C. 

"Complement  Fixation  in  Pulmonary  Tuberculosis,"  Radcliffe,  S.  A.  D.,  Jour,  of  Hygiene, 
1915,  vol  xv,  No.  i,  pp.  36-50. 

"Complement  Fixation  in  Pertussis,"  Olmstead  and  Tuttiriger,  Archives  Int.  Med.,  1915,  vol. 
xvi,  No.  i. 

"The  Separation  of  Protozoan  Species  by  Means  of  Immunity  Reactions,"  Coca,  A.  F.,  Zeitsch. 
fur  Immunitatsforschung  und  exper.  Therapie,  1912,  Jan.  17. 


CHAPTER  IX 
IMMUNITY 

Immunity  from  disease  is  dependent  upon  innumerable  extrinsic  and  intrinsic 
factors,  many  of  which  are  not  constants.  Hence  immunity  is  seldom  if  ever 
absolute,  it  is  relative  or  conditional  or  partial;  it  varies  in  different  geo- 
graphical locations,  in  different  individuals  under  similar  circumstances;  in 
each  of  us  it  ebbs  and  flows  like  the  tide,  changes  from  hour  to  hour  and  con- 
spicuously varies  at  different  age  periods  of  life — in  infancy,  youth  and  old  age. 

We  are  more  or  less  familiar  with  some  of  the  extrinsic  and  intrinsic  factors 
that  establish  and  destroy  immunity  and  there  are  others  of  which  we  know 
nothing. 

Of  all  the  ailments  afflicting  man  half,  or  more,  are  infections — diseases  in 
which  the  body  is  assaulted  by  microorganisms  or  their  products. 

The  two  most  important  factors  in  every  case  of  infectious  disease  are  first, 
the  animal  afflicted,  and  second,  the  offending  microorganism.  For  infection  to 
develop  each  of  these  factors  must  fulfill  certain  requirements  and  we  may,  for 
convenience  of  study  at  first,  consider  them  separately. 

PROPERTIES  PECULIAR  TO  PATHOGENIC  BACTERIA 

Bacteria  potentially  capable  of  causing  disease  are  those  that  produce  aggres- 
sin,  endotoxin,  ectotoxin — one,  two,  or  all  of  which  can  propagate  in  the  host. 

Aggressin. — Having  inoculated  animals  with  the  typhoid  bacillus  and  so 
produced  a  serous  exudate,  Bail  collected  this  exudate,  freed  it  of  bacteria  by 
centrifugalization  and  mixed  a  small  quantity  of  it  with  a  sublethal  dose  of 
typhoid  bacilli.  By  injecting  this  mixture  into  experimental  animals  he  pro- 
duced death. 

Bail's  explanation  of  this  phenomenon  is  substantially  as  follows:  Pathogenic 
bacteria,  upon  entering  a  host,  elaborate  a  substance  that  protects  the  bacterium 
and  assures  its  development  by  paralyzing  or  destroying  the  bacteriocidal  sub- 
stances of  the  host  which  are  attracted  to  the  atrium  of  infection,  especially 
those  substances  that  effect  phagocytosis. 

This  bacterial  emanation,  capable  of  inhibiting  phagocytosis,  he  named 
aggressin. 

Sterile  serous  exudates  containing  aggressin  are  of  themselves  poisonous; 
heating  for  i  hour  at  6o°C.  does  not  impair  their  toxicity;  by  repeated  injections 
of  small  quantities  of  them  a  specific  immunity  to  the  aggressin  and  bacterium 
producing  it  can  be  conferred  upon  experimental  animals. 

Endotoxin.— By  various  experiments,  not  necessary  to  describe  here,  one 
can  demonstrate  that  all  pathogenic  bacteria  produce  a  water-  and  serum-solu- 
ble substance  that  is  retained  within  the  bacterium  until  it  begins  to  involute  or 

259 


260  MEDICAL  BACTERIOLOGY 

dies  or  disintegrates  and  is  then  liberated.  Solutions  of  this  substance,  obtained 
by  extracting  dead  bacteria  with  water  or  salt  solution,  when  injected  into 
animals  produces  some  or  all  of  the  pathological  changes  and  obvious  signs  of 
disease  that  are  commonly  produced  by  infection  with  the  species  of  bacteria 
from  which  the  soluble  extract  was  obtained — this  substance  is  referred  to  as 
intracellular  toxin  or  endotoxin. 

Endotoxins  obtained  from  different  species  of  bacteria  differ  in  quantity, 
quality  and  resistance  to  heat.  They  all  withstand  heating  to  55°C.  for  J^ 
hour,  many  withstand  exposures  of  several  hours  to  6o°C.,  7o°C.,  8o°C.  or  even 
90°C.;  a  few.  like  tuberculin,  withstand  heating  to  no°C.  for  several  hours. 

Repeated  ascending  doses  of  endotoxin  obtained  from  certain  species  of 
bacteria  confer  immunity  to  the  species  from  which  the  endotoxin  was  ob- 
tained— a  specific  immunity. 

There  is  a  close  similarity  in  many  respects  between  aggressin  and  endotoxin, 
in  fact,  Wassermann  and  Citron  believe  that  what  Bail  described  as  aggressin 
is  in  reality  endotoxin. 

As  a  general  rule,  to  which  there  are  exceptions,  the  virulence  of  a  bacterium 
is  proportional  to  the  nocuous  power  of  its  endotoxin. 

Ectotoxin. — A  few  of  the  pathogenic  bacteria  throughout  their  active  life 
emanate  highly  poisonous  soluble  substances  that  are  absorbed  by  the  tissue 
or  medium  upon  which  the  bacteria  are  located.  This  poisonous  emanation  is 
referred  to  as  extracellular  toxin  or  ectotoxin.  It  is  water-soluble  and  serum- 
soluble.  The  pathogenicity  and  virulence  of  bacteria  that  produce  ecto- 
toxin are  largely  dependent  upon  and  proportional  to  the  nocuousness  of  the 
emanation. 

The  ectotoxins  as  a  whole  are  much  less  resistant  to  heat  than  endotoxins, 
the  most  stable  being  destroyed  at  ioo°C.  in  a  few  minutes. 

The  ectotoxins  produced  by  different  species  of  bacteria  differ  widely  in  their 
affinity  for  and  effect  upon  tissue  cells  in  vivo. 

Repeated  injections  of  ectotoxin  confer  a  relatively  high  degree  of  immunity 
to  the  specific  ectotoxin  injected  and  a  slight  immunity  to  the  species  of  bacteria 
from  whence  it  came. 

If  we  study  different  strains  of  a  single  species  or  successive  generations  of  a 
single  strain,  it  soon  becomes  evident  that  bacteria  vary  in  their  toxin  produc- 
tion; this  is  especially  true  of  ectotoxin  production.  Slight  alterations  in  tem- 
perature, aerobiosis,  hydrogen  ion  concentration,  acidity  or  alkalinity  of 
environment,  and  pabulum,  cause  sudden,  marked  increases  and  decreases  in 
ectotoxin  production. 

At  times,  for  no  apparent  reason,  cultures  cease  producing  ectotoxin,  the 
cessation  being  temporary  or  permanent.  There  are  conspicuous  exceptions  as 
in  the  case  of  the  diphtheria  bacillus  cultivated  by  Williams  which  has  remained 
practically  constant  in  its  toxin  production  for  years. 

VIRULENCE 
The  virulence  of  bacteria  is  subject  to  alteration  by  many  factors,  some  of 


IMMUNITY  26l 

which  are  known.  Alterations  in  virulence  may  be  temporary  or  permanent; 
both  attenuation  and  exaltation  of  virulence  occurs. 

Pasteur  showed  that  passage  of  rabies  virus  (which  we  assume  is  bacterial) 
through  successive  rabbits  produces  progressive  alteration  of  its  character  and 
virulence  up  to  a  certain  point,  beyond  which  it  remains  constant,  no  matter 
how  many  times  it  is  passed  from  rabbit  to  rabbit. 

During  its  transitional  stage,  modification  of  the  virus  is  shown  by  a  gradual 
shortening  of  the  period  of  incubation  following  inoculation,  and  curtailment  of 
the  stage  of  excitement  when  symptoms  develop.  When  alterations  in  the 
character  of  the  virus  cease  subsequent  inoculations  of  rabbits  are  followed  by 
a  very  uniform  short  period  of  incubation  (5  to  6  days);  the  clinical  signs  of 
disease  are  those  of  a  rapidly  progressive  and  fatal  paralysis  without  a  preceding 
stage  of  excitement.  Injected  into  animals  other  than  the  rabbit,  or  into  man, 
fixed  virus  does  not  produce  rabies — it  confers  immunity  to  rabies. 

These  observations  may  be  summarized  as  follows: 

First. — Passage  of  rabies  virus  from  rabbit  to  rabbit  produces  alterations  in 
it  up  to  a  certain  point  beyond  which  it  remains  constant  in  character  and  is 
referred  to  as  "fixed  virus." 

Second. — Passage  of  rabies  virus  from  rabbit  to  rabbit  increases  its  virulence 
for  rabbits  and  decreases  its  virulence  for  other  animals. 

If  anthrax  bacilli,  primarily  virulent  to  sheep,  are  cultivated  at  a  constant 
temperature  of  42°C.  to  43°C.,  they  gradually  lose  their  virulence  for  animals  in 
general.  It  is  possible  to  carry  this  attenuation  to  any  desired  degree,  and  also 
possible  to  arrest  it  and  thereafter  maintain  the  strain  at  any  desired  virulence 
as  is  done  in  the  preparation  of  anthrax  vaccine  (page  222). 

In  fixed  rabies  virus  and  anthrax  vaccine  we  have  two  examples  of  perma- 
nent modification  of  virulence  deliberately  effected;  the  former  being  intensified 
for  one  species  and  attenuated  for  other  species;  the  latter  being  attenuated  for 
animals  in  general. 

Among  the  various  pathogenic  species  of  bacteria  strains  are  encountered 
that,  from  unknown  causes,  or  as  a  result  of  prolonged  cultivation  on  artificial 
media,  have  partially  or  entirely,  and  presumably,  permanently  lost  virulence 
as  in  the  case  of  the  avirulent  tubercle  bacillus  employed  by  Von  Ruck  to 
produce  tuberculin. 

From  the  evidence  we  have  it  seems  highly  probable  that  permanent  altera- 
tions in  virulence,  both  exaltation  and  attenuation,  occur,  but  there  is  no  criter- 
ion by  which  one  can  distinguish  permanent  from  temporary  alterations  of 
virulence.  Instances  of  presumably  permanently  attenuated  organisms  proving 
virulent  are  not  lacking. 

FLUCTUATIONS  IN  VIRULENCE 

Much  more  commonly  met  with  than  permanent  alterations  of  virulence 
are  temporary  alterations  of  brief  duration.  It  is  constantly  observed  in  prac- 
tice that  most  bacteria  manifest  their  greatest  virulence  when  passed  directly 
from  animal  to  animal,  especially  when  passed  from  one  animal  to  another  of 


262  MEDICAL  BACTERIOLOGY 

the  same  species,  and  show  lessened  virulence  after  periods  of  vegetative  exist- 
ence, particularly  so  if  some  of  the  conditions  of  environment  have  been  detri- 
mental to  their  development.  Thus  streptococci  are  observed  which  when 
isolated  from  a  lesion  of  man  are  very  virulent  for  rabbits  and  perhaps  also  viru- 
lent for  guinea-pigs  but  after  several  weeks  or  months  of  cultivation  on  artificial 
media  become  avirulent  for  guinea-pigs  and  only  slightly  virulent  for  rabbits. 
If  a  large  dose  of  such  attenuated  streptococci  is  injected  into  a  rabbit,  disease 
develops  and  organisms  from  the  afflicted  rabbit  directly  transferred  to  a  second 
rabbit  show  increased  virulence,  and  by  several  passages  from  rabbit  to  rabbit 
the  original  high  degree  of  virulence  may  be  restored. 

Exactly  what  change  occurs  in  bacteria  that  results  in  alteration  of  virulence 
is  not  clear,  but  some  of  the  obvious  changes  manifested  by  some  bacteria  that 
have  acquired  immunity  are  suggestive. 

ACQUIRED  IMMUNITY  OF  BACTERIA 

Normally  rat  blood  is  capable  of  destroying  large  numbers  of  anthrax  bacilli. 
If  a  minute  quantity  of  rat  serum  is  added  to  a  broth  culture  of  anthrax  bacilil 
and  gradually  increasing  amounts  are  added  to  subcultures,  eventually  subcul- 
tures will  grow  abundantly  in  pure  rat  serum.  This  immunity  is  inherited  by 
subcultures  grown  in  plain  broth  and  it  is  specific. 

Danysz  demonstrated  that  anthrax  bacilli  immune  to  rat  serum  have 
developed  a  mucous  sheath  or  capsule.  This  mucous  capsule  unites  with  the 
germicidal  substance  in  rat  serum  and  makes  it  innocuous  for  anthrax  bacilli. 

Metchnikoff,  Trommsdorf  and  others  have  shown  that  under  analogous 
conditions  streptococci,  tubercle  bacilli,  the  typhoid  bacillus  and  cholera  vibrio 
develop  a  mucous  capsule  which  immunizes  them  against  substances  normally 
germicidal,  including  arsenic  and  bichloride  of  mercury;  that  many  of  these 
acquired  immunities  become  permanent  and  are  transmitted  to  future  genera- 
tions, that  some,  at  least,  of  the  bacteria  retain  all  the  faculties  and  virulence 
after  acquiring  immunity  that  they  possessed  before.  Furthermore,  the 
acquired  immunity  of  bacteria  is  specific,  i.e.,  a  bacterium  that  has  acquired 
immunity  to  the  serum  of  one  species  of  animals  is  just  as  susceptible  to  the  sera 
of  other  species  of  animals  as  it  was  before;  anthrax  bacilli  that  have  acquired 
immunity  to  formaldehyde  are  just  as  susceptible  to  mercury  and  phenol  as 
anthrax  bacilli  in  general. 

In  acquiring  active,  specific  immunity,  some  microorganisms,  by  the  same 
process,  acquire  other  new  faculties.  Effront  has  shown  that  when  brewers' 
yeast  is  accustomed  to  living  in  media  containing  quantities  of  hydrofluoric 
acid  germicidal  to  other  yeasts,  its  power  to  produce  alcohol  is  augmented  and 
its  susceptibility  to  chemical  germicides  other  than  hydrofluoric  acid  increased. 

Although  there  is  not  sufficient  data  upon  which  to  base  broad  generaliza- 
tions, collected  observations  indicate  that  as  a  rule  vegetative  existence  lessens 
and  parasitic  existence  increases  the  virulence  of  bacteria;  that  when  changed 
from  a  favorable  to  an  unfavorable  environment  bacteria  either  lose  some  of 
their  faculties  or  acquire  new;  virulence  being  exalted  in  some  instances  and 


IMMUNITY  263 

attenuated  in  most;  generally,  when  existence  in  an  unfavorable  medium  is  of 
short  duration  changes  so  effected  are  temporary,  as  manifested  by  a  resumption 
of  primary  virulence  when  restored  to  a  favorable  environment;  prolonged 
cultivation  of  bacteria  in  an  unfavorable  medium  tends  to  produce  permanent 
changes  in  them  which  become  hereditary. 

INFECTIOUSNESS  OF  BACTERIA 

If  we  employ  the  word  infection  to  indicate  not  merely  the  lodgment  of 
bacteria  upon  or  in  tissue,  but  the  subsequent  multiplication  of  them  with 
injury  to  the  host,  important  differences  in  the  infectious  power  of  different 
species  becomes  apparent  together  with  definite  facts  that  determine  in  a  given 
case  whether  the  lodgment  of  bacteria  upon  or  in  tissue  can  or  cannot  initiate 
infection. 

Webb  has  shown  that  the  injection  of  one,  two  or  three  tubercle  bacilli 
never  produces  tuberculosis;  but  the  injection  of  10  or  more  of  the  most  virulent 
tubercle  bacilli  regularly  produces  tuberculosis.  If  tubercle  bacilli  only  half  as 
virulent  are  employed,  however,  the  minimum  number  required  to  produce 
infection  is  more  than  20.  Webb  made  these  observations  when  experimenting 
with  monkeys. 

There  is  a  great  mass  of  evidence  indicating  that  the  probability  of  infection 
following  the  lodgment  of  bacteria  is  in  proportion  to  their  number.  It  is 
highly  improbable  that  infection  ever  follows  the  lodgment  of  one  or  several 
bacteria. 

Certain  organisms  manifest  a  predilection  for  certain  tissues,  thus  a  quantity 
of  rabies  virus  which  will  regularly  produce  disease  when  injected  beneath  the 
dura,  very  rarely  causes  infection  when  injected  subcutaneously  or  into  a  mus- 
cle. Rapidly  fatal,  disseminated  tuberculosis,  as  well  as  the  chronic  pulmonary 
type,  rarely  shows  involvement  of  muscles. 

The  infectiousness  of  some  bacteria  is  influenced  by  association  or  coincident 
lodgment  with  other  species  of  bacteria  and  medicinal  agents.  When  inunctions 
of  mercury  are  applied  at  the  point  of  entry  within  several  hours  after  the  lodg- 
ment of  the  treponema  pallidum  infection  is  frequently  prevented.  If  quinine 
enters  the  body  at  the  time  tetanus  spores  gain  lodgment,  or  shortly  afterward/ 
the  development  of  infection  is  favored.  Though  the  influences  causing  it  are 
largely  undeterminate,  it  is  worthy  of  note  in  this  connection,  that  the  existence 
of  diphtheria  seems  to  predispose  to  scarlet  fever;  that  diphtheria,  superimposed 
on  Vincent's  angina,  is  unusually  virulent;  that  the  existence  of  gonorrhea  favors 
secondary  infection  of  the  urethra  by  the  pseudodiphtheria  bacillus. 

RESISTANCE  OF  MAN  TO  INFECTION 

Resistance  to  infection  is  both  general  and  specific.  Man  naturally  possesses 
certain  forces  that  tend  to  destroy  any  species  of  bacteria  that  gain  lodgment 
upon  or  in  the  body.  Many  bacteria  are  destroyed  by  the  hydrochloric  acid  of 
the  stomach.  Most  pathogenic  bacteria  cannot  cause  disease  until  they  have 


264  MEDICAL  BACTERIOLOGY 

entered  the  body  through  the  skin  or  mucous  membrane  and  in  a  physiologically 
perfect  state,  the  skin  and  mucous  membranes  are  impervious  to  these  organisms. 

The  blood  contains  fluids  and  solids  that  attack  and  attempt  to  remove  or 
destroy  any  bacteria  they  meet.  Most,  if  not  all,  the  tissue  cells  of  the  body 
in  a  physiologically  normal  state  to  some  degree  resist  invasion  by  bacteria. 

In  addition  to  these  natural,  general,  immunizing  forces,  man  acquires 
other,  often  stronger,  specific  resistance  forces  that  act  upon  a  single  species  of 
bacteria  and  are  developed  as  a  result  of  repeated  trivial  infections  by  that 
species  or  one  acute  infection  by  that  species  or  treatment  with  a  vaccine. 

The  effectiveness  of  both  general  and  specific  immunizing  forces  fluctuates 
with  changes  in  the  body  state.  It  is  proportional  to  the  physiological  condition 
and  vigor  of  the  body  as  a  whole  and  to  that  of  its  various  organs.  When  any 
of  the  body  as  a  whole  and  to  that  of  its  various  organs.  When  any  of  the  body 
functions  are  impaired,  arrested  or  destroyed,  an  immediate  decline  in  resistance 
to  infection  occurs.  Indigestion,  insomnia  and  constipation  all  lessen  resistance. 
Fatigue,  mental  or  physical,  lessens  resistance.  Of  two  extremities  otherwise 
equal,  differing  in  that  one  has  been  bruised  or  crushed — the  injured  extremity 
is  most  vulnerable  to  infection. 

There  is  so  slight  a  difference  between  the  infectiousness  of  many  bacteria 
and  the  corresponding  resistance  of  the  body,  that  frequently,  the  result  of 
lodgment  of  pathogenic  bacteria  upon  or  in  the  body,  whether  they  cause 
disease  or  are  resisted,  is  determined  by  the  state  of  the  body  at  that  time — 
exhausted  or  not  exhausted,  well  nourished  or  poorly  nourished,  constipated  or 
not. 

Immunity,  both  natural  and  acquired,  to  some  organisms  is  so  nearly  abso- 
lute that  infection  seldom  follows  exposure  to  it  even  when  vitality  is  low  and 
physiological  activity  and  coordination  poor. 

MECHANISM  OF  IMMUNITY 

Metchnikoff  has  clearly  shown  that  many  of  the  tissue  cells  and  especially 
endothelial  cells  and  polymorphonuclear  leucocytes  elaborate  a  ferment-like 
substance  Cytase  which  destroys  or  facilitates  the  destruction  of  bacteria. 
Wright  disclosed  the  presence  in  blood  serum  and  fluid  expressed  from  muscular 
and  connective  tissues  of  a  substance  called  opsonin  which  facilitates  ingestion 
and  digestion  of  bacteria  by  phagocytic  cells.  Pfeiffer  discovered  that  under 
certain  conditions  peritoneal  exudate  causes  a  granular  degeneration  of  the 
cholera  vibrio. 

PFEIFFER'S  PHENOMENON 

Pfeiffer's  test  (page  123)  is  explained,  on  experimental  evidence,  by  Metchni- 
koff as  follows: 

Immediately  after  the  injection  of  living  cholera  vibriones  into  the  peritoneal 
cavity  of  a  guinea-pig,  disintegration  of  the  phagocytic  cells  surrounding  the 
organisms  occurs — Phagolysis — with  a  consequent  liberation  of  their  cytase. 
The  cytase  liberated  by  the  cells  of  susceptible  animals  is  not  able  to  injure 


IMMUNITY  265 

the  cholera  vibriones  and  consequently  they  retain  their  original  morphology 
and  motility  and  multiply. 

The  phagocytic  cells  of  immunized  guinea-pigs  contain  a  more  potent  cytase 
and  when  this  is  liberated  by  phagolysis  it  causes  the  granular  degeneration  and 
loss  of  motility  and  arrest  of  propagation  observed. 

Bordet  observed  that  when  a  sublethal  dose  of  streptococci  is  injected  into 
the  peritoneal  cavity  of  a  guinea-pig  a  high  leucocytosis  promptly  occurs  and  the 
cocci  are  all  taken  up  and  destroyed  by  the  leucocytes.  If  a  lethal  dose  is 
injected  phagocytosis  occurs  as  before  but  some  cocci  escape  and  these  produce  a 
generation  more  resistant  to  phagocytosis  so  that,  even  though  leucocytosis 
continues  high  a  greater  proportion  of  this  second  generation  escapes  destruc- 
tion, the  cocci  can  multiply  faster  than  the  phagocytes  can  destroy  them  and 
general  dissemination  results. 


FIG.  43. — (Citron.}  FIG.  44. — (Citron.) 

In  his  original  investigations  of  phagocytosis  Wright  devised  a  method  of 
studying  changes  in  it,  now  known  as  the  opsonic  index  test.  The  test  is 
made  as  follows: 

Blood  is  collected  in  a  capillary  tube  from  the  patient,  the  tube  is  sealed  and 
put  aside  to  clot  (see  Fig.  43).  Several  loopsful  of  bacteria  are  removed 
from  a  24-  or  48-hour-old  culture  on  solid  media  and  emulsified  with  normal 
salt  solution;  this  suspension  is  drawn  into  a  tube  and  centrifugalized  for  a 
minute  to  throw  down  clumps;  the  supernatant  suspension  is  then  removed. 
Blood  is  collected  from  the  patient's  finger  in  a  capillary  tube,  half  rilled  with  i 
per  cent,  sodium  citrate  in  normal  salt  solution.  The  tube  is  shaken  to  mix 
the  citrate  solution  and  blood,  sealed,  and  centrifugalized  until  a  distinct 
layer  of  white  cells  is  seen  upon  the  top  of  the  rods.  The  white  cells  are 
pipetted  off  and  placed  in  a  sterile  watch  crystal.  The  patient's  serum  is 
poured  from  the  clot  into  another  sterile  watch  crystal  and  the  bacterial  sus- 
pension is  placed  in  a  third  watch  crystal. 

The  bulb  of  a  mixing  pipette  is  compressed  and  one  volume  of  white  cells 
drawn  in  by  partially  releasing  the  bulb ;  the  bulb  is  further  released  so  that  the 
cells  retreat  from  the  tip ;  then  a  volume  of  bacteria  is  drawn  into  the  tube  and  a 
bubble  of  air  admitted;  finally  a  volume  of  patient's  serum  is  taken  into  the  tube. 


266 


MEDICAL  BACTERIOLOGY 


The  contents  of  the  tube  are  then  thoroughly  mixed  by  ejecting  them  into  a 
sterile  watch  crystal  and  drawing  them  back  into  the  tube  several  times.  When 
mixed  the  fluid  is  drawn  to  the  middle  of  the  tube,  the  ends  sealed  and  an  identi- 
fication mark  placed  on  the  tube.  The  tube  is  then  incubated  at  body  tempera- 
ture for  15  minutes  in  an  incubator  that  will  permit  fre- 
quent rotation  of  the  tube  as  this  is  necessary  (see  Fig.  45). 
At  the  same  time  that  blood  is  drawn  from  the  pa- 
tient to  obtain  serum,  blood  is  also  collected  for  the  same 
purpose  from  several  healthy  persons  and  the  normal  sera 
is  pooled  (mixed). 

A  second  mixing  pipette  is  loaded  with  equal  volumes 
of  cells,  bacteria  and  pooled  serum  and  treated  exactly 
the  same  as  the  tubes  containing  patient's  serum. 

When  the  period  of  incubation  is  up,  the  tube  contain- 
ing patient's  serum  is  emptied  on  a  clean  slide,  one  drop  of 
the  fluid  is  transferred  to  each  of  several  other  slides  and 
these  drops  are  spread  in  a  thin  film. 

The  slides  are  fixed  and  stained.  Staining  with  Leish- 
man's  blood  stain  is  the  common  method  but  primary 
fixation  with  methyl  alcohol  for  i  minute  followed  by 
staining  with  dilute  fuchsin  for  a  minute  gives  a  more  dis- 
tinct field.  The  slides  so  prepared  are  examined  with  the 
oil  immersion  lens.  The  number  of  bacteria  within  each 
leucocyte  is  recorded  and  when  100  leucocytes  have  been 
examined  the  average  number  of  bacteria  per  leucocyte 
is  computed;  this  number  is  the  patient's  opsonic  content. 
By  examining  in  the  same  way  the  contents  of  the 
second  tube  the  normal  opsonic  content  is  determined. 

The  patient's  opsonic  content,  divided  by  the  normal 
opsonic  content,  equals  the  patient's  opsonic  index. 

Using  this  test  Wright  discovered  the  following  facts. 
When  washed  free  of  blood  serum    leucotyes  almost  en- 
tirely lose  their  phagocytic  power;  in   other  words,  there 
Air  bubble  *s  some  substance  in  blood  serum  which  does  not  destroy 
bacteria  but  acts  on  them  so  as  to  facilitate  their  ingestion 
and  digestion  by  leucocytes;  to  this   substance   he   gave 
the  name  opsonin.     The  quantity  of  potency  of  opsonin 
varies;  it  is  low  when  immunity  is  slight  and  high  when 
immunity   is    strong.     There    is     natural    opsonin — that 
which    is    present    throughout    life    regardless    of    infection — and    acquired 
opsonin,  developed  as  a  result  of  infection  or  vaccination. 

There  may  be  both  thermolabile  and  thermostabile  opsonins  present  at  the 
same  time  in  a  given  animal's  serum. 

Opsonin  seems  to  be  entirely  specific  in  nature — in  an  animal  it  may  be 
below  normal  for  one  organism  and  normal  for  other  organisms;  in  vitro,  the 


Mar 


Leucocytes 

Air  bubble 
Bacilli 


Serum 


FIG.  45. — (Citron.} 


IMMUNITY  267 

opsonin  for  one  species  of  bacteria  may  be  exhausted  from  a  serum  without 
lessening  in  any  way  the  opsonic  content  for  other  species  of  bacteria. 

When  a  patient  suffers  infection  his  opsonic  content  is  primarily  below 
normal  for  the  infecting  organism;  if  the  disease  progresses  to  a  favorable  ter- 
mination the  opsonic  content  of  the  patient's  serum,  for  the  infecting  organism, 
shows  a  progressive  increase  from  shortly  before  or  after  the  disease  reaches  its 
fastigium  until  recovery,  and  it  may  be  distinctly  above  normal  in  the  period  of 
convalescence — continuing  so  for  weeks  or  months. 

Vaccines  produce  a  similar  effect.  Following  the  administration  of  a  dose 
of  bacteria  or  bacterial  extractives  there  is  a  sharp  fall  in  the  opsonic  content, 
the  degree  and  duration  of  which  is  proportioned  to  the  dose;  this  period  of 
depression  is  known  as  the  "negative  phase." 

An  overdose  (poisonous  dose)  of  vaccine  frequently  induces  a  negative  phase 
of  long  duration  followed  by  a  slow  return  to  normal.  In  a  few  favorable 
instances  an  overdose  is  followed  by  a  marked  fall  of  short  duration  superseded 
by  a  rapid  elevation  above  normal.  Therapeutic  or  immunizing  doses  produce 
a  slight  negative  phase  of  12  to  72  hours  duration  followed  by  a  gradual  rise, 
2  to  12  days  in  duration,  averaging  a  distinct  elevation  of  the  opsonic  content 
above  that  which  existed  prior  to  treatment. 

Slight  fluctuations  in  the  opsonic  content  for  the  infecting  organism  during 
the  course  of  a  disease  frequently  are  not  associated  with  discernible  changes  in 
physical  signs  but  marked  fluctuations  usually  are,  descent  of  the  opsonic  con- 
tent being  associated  with  physical  signs  of  ill  omen  and  ascent  concomitant 
with  favorable  changes  in  physical  signs. 

OTHER  PROTECTIVE  SUBSTANCES  IN  BLOOD  SERUM 

Previous  descriptions  of  agglutinins,  lysins  and  antitoxins  explain  in  part  the 
stimuli  causing  production  of  such  of  these  as  are  acquired,  their  degrees  of 
specificity  and  mode  of  action  and  their  more  important  physical  properties 
and  effects. 

There  are  specific  agglutinins,  lysins,  precipitins  and  antitoxins  present  in 
the  blood  serum  prior  to  infection  or  inoculation.  These  form  part  of  the 
natural  protective  armament  against  infection.  In  general,  primary  anti- 
bodies are  less  abundant  and  less  effective  than  those  formed  as  a  result  of 
infection  or  inoculation  (acquired  antibodies)  but  are  identical  in  nature  and 
action. 

There  are  notable  exceptions  to  this  rule  as  evidenced  by  immunity  to 
diphtheria.  Schick  and  Park  have  shown  that  about  75  per  cent,  of  all  children 
are  immune  to  diphtheria  during  the  first  3  months  of  life;  during  the  remainder 
of  the  first  2  years  only  50  per  cent,  are  immune;  the  percentage  of  children 
immune  to  diphtheria  gradually  increases  from  the  second  year  of  life  up  to 
maturity,  at  which  time  about  80  per  cent,  are  immune  even  though  they  have 
not  had  the  disease.  Evidence  indicates  that  this  immunity  is  largely,  if  not 
entirely,  due  to  the  presence  of  antitoxin  in  the  blood.  Here  then  is  an  example 
of  natural  antitoxin  present  in  sufficient  quantity  to  protect  against  infection; 


268  MEDICAL  BACTERIOLOGY 

antitoxin  received  from  the  mother  before  birth  and  lost  during  the  first  3 
months  of  life  but  gradually  replaced  by  the  possessor's  own  cells  and  apparently 
without  the  stimulus  of  infection. 

Rabbits  normally  possess  agglutinins  for  pneumococci  but  after  inoculation 
with  sublethal  doses  of  pneumococci  they  develop  more  abundant,  and,  there- 
fore, more  effective  pneumococcus — agglutinin.  Bull  has  found  that  pneumo- 
cocci injected  intravenously  soon  disappear  from  the  circulation  as  a  result  of 
agglutination.  If  sufficient  numbers  are  injected  the  natural  agglutinin  is 
exhausted  and  cocci  escaping  destruction  lodge  in  tissue  and  multiply.  After  a 
time,  stimulated  by  the  infection,  more  agglutinin  is  produced  and  coincident 
with  the  appearance  of  acquired  agglutinin  recession  of  the  disease  begins. 

In  addition  to  the  antibodies  that  have  been  demonstrated  and  described, 
there  are  others,  possibly  of  equal  importance  in  the  establishment  of  immunity, 
that  are  still  obscure.  Sclavo's  serum  which  unquestionably  possesses  immuniz- 
ing power  contains  little  or  no  opsonin,  complement,  lysin,  agglutinin,  antitoxin, 
precipitin  or  cytase.  It  is  observed  at  times  that  serum  containing  no  demon- 
strable antibodies  from  an  immune  animal  will  prevent  the  multiplication  of 
bacteria  without  otherwise  affecting  them. 

THEORIES  OF  IMMUNITY 

After  more  than  a  quarter  of  a  century  of  research  and  observation  of  the 
phenomena  accompanying  infection  and  immunity  in  various  animals  from  the 
simple  unicellular  forms  up  to  and  including  man,  Metchnikoff  concluded  that 
invading  organisms  exert  varying  degrees  of  chemiotaxis;  some  a  negative 
chemiotaxis,  others  a  slight  positive  and  some  a  strong  positive  chemiotaxis  on 
the  phagocytic  cells  of  the  host.  In  the  mammals  this  action  is  exerted  on 
certain  body  cells  and  is  most  strikingly  exemplified  by  the  chemiotaxis  of 
some  bacteria  for  leucocytes. 

He  believed  the  production  of  cytase  and  other  antibodies,  above  and  in 
addition  to  what  normally  exists,  depends  upon  two  factors:  stimulation  of  the 
cells  of  the  host  by  the  invading  organism;  and  an  increased  production  of 
cytase,  agglutinin,  lysin, -etc.,  by  the  cells  stimulated,  the  increase  being  in 
quantity  or  quality  or  both. 

Metchnikoff  considered  digestion  of  bacteria  within  the  phagocytic  cells  of 
the  body  the  ultimate  and  also  the  essential  process  in  ridding  the  body  of 
bacteria.  He  believed  excretory  organs  in  a  normal  condition  never  excrete 
bacteria.  He  assumed  that  the  action  of  antibodies  in  the  blood  serum  on 
bacteria  facilitates  their  ingestion  and  digestion  by  phagocytic  cells  but  con- 
sidered bacteriolysis  an  exclusively  intracellular  phenomenon. 

The  observations  of  some  of  his  contemporaries  and  followers  have  amply, 
confirmed  some  of  Metchnikoff 's  views  and  discredited  others.  It  is  now  recog- 
nized that  the  establishment  and  maintenance  of  active  immunity  and  the  con- 
quest of  infection  is  a  matter  of  cellular  activity;  in  some  cases  the  activity  of 
phagocytic  cells  and  in  other  cases  the  activity  of  cells  (not  phagocytes)  that 
emanate  antibodies  which  flow  in  the  blood  serum  and  attack,  arrest  or  destroy 
bacteria  or  neutralize  their  toxins. 


IMMUNITY 


269 


EHRLICH'S  THEORY 

From  his  studies  of  the  production  of  antitoxins,  agglutinins,  lysins  and  pre- 
cipitins  and  their  action  both  in  vivo  and  in  -vitro  Ehrlich  evolved  a  theory  of 
immunity  commonly  referred  to  as  "Ehrlich's  Side-Chain  Theory,"  which  may 
be  briefly  summarized  as  follows: 

The  digestion  of  food  in  the  alimentary  canal  is  not  an  adequate  preparation 
of  it  for  assimilation  by  various  tissue  cells,  hence  each  tissue  cell  of  the  body 
must  select  and  draw  to  itself  certain  substances  in  the  blood  serum  and  further 
digest  them  for  its  nutrition;  and  reject  other  substances  in  the  serum  not 
appropriate  to  its  needs. 

Tissue  cells  possess  numerous  side  chains,  each  having  an  affinity  for  a 
particular  substance  needed  by  the  cell  for  its  nutrition. 

In  conformity  with  a  general  law  of  nature  when  these  side  chains  or  receptors 
are  exhausted  and  when  the  requirements  of  the  cell  increase,  a  superabundance 
of  new  receptors  is  produced.  Receptors  produced  in  excess  of  the  cell's  needs 
are  cast  off  and  circulate  in  the  blood  serum.  Receptors  in  the  blood  serum 
manifest  the  same  specific  affinities  and  unite  with  the  same  substances  as  when 
attached  to  cells,  precipitating,  agglutinating,  neutralizing  or  disintegrating 
them.  This  may  be  illustrated  by  the  following  diagrams  (Fig.  46.): 


FIG.  46. 

I.  Represents  a  cell  with  two  different  kinds  of  receptors.     C,  cell;  A,  recep- 
tors with  affinity  for  one  substance;  B,  receptors  for  another  substance. 

II.  Represents   the   same   cell  after   stimulation  producing   an   increased 
number  of  receptors. 

III.  Some  of  the  excess  receptors  cast  off  by  the  cell. 

IV.  Represents  poisonous  substance  or  food  with  which  receptors  A  would 
unite. 

Appetite,  digestion  within  the  stomach  and  intestines  and  absorption  from 
these  organs  is  for  alimentation;  some  substances  devoid  of  nutritive  properties 
are  not  assimilated  when  taken  into  the  alimentary  canal,  but  pass  through  it 
without  affecting  the  body. 

One  may  possess  an  appetite  for  that  which  is  poison  and  not  food;  the 
stomach  and  intestines  may  digest  and  pass  into  the  circulation  some  noxious 


270  MEDICAL  BACTERIOLOGY 

substances.  The  same  is  true  of  cellular  digestion.  Some  cells  possess  recep- 
tors which  manifest  an  equal  or  greater  affinity  for  certain  poisons  than  for 
food ;  if  the  blood  serum  brings  such  poison  to  a  cell  so  equipped  it  is  assimilated 
and  as  a  result  one  of  two  possibilities  result:  the  cell  dies,  or  it  is  injured,  the 
receptors  uniting  with  the  poison  exhausted,  the  cell  temporarily  arrested  in 
its  activities;  the  arrest  stimulates  greater  activity  on  the  part  of  the  cell, 
and  as  it  recuperates  the  ceil  not  only  reproduces  the  destroyed  parts,  but 
forms  many  more  receptors  than  were  exhausted,  so  many,  that  some 
are  cast  off  and  taken  up  by  the  blood  serum.  If  the  blood  serum  receives 
more  of  the  poison,  the  free  receptors  in  the  serum  unite  with  it  and  precipitate, 
agglutinate,  neutralize  or  disintegrate  it,  so  preventing  the  poison  from  reach- 
ing and  injuring  the  cell — the  cell  is  immune  so  long  as  the  serum  contains 
free  receptors. 

Ehrlich  showed  that  these  receptors  or  antibodies  are  divisible  into  three 
groups:  receptors  of  the  first  order,  receptors  of  the  second  order  and  receptors 
of  the  third  order.  There  are  certain  properties  common  to  all  three  and  others 
peculiar  to  each  group.  All  are  products  of  cellular  activity,  all  occur  in  blood 
serum,  all  are  to  a  degree  specific  in  action  uniting  with  a  single  substance,  all 
may  be  increased  up  to  a  certain  point  by  stimulation  of  the  cells  producing 
them. 

Receptors  of  the  first  order  include  the  antitoxins.  In  the  blood  serum  they 
are  almost  or  entirely  confined  to  the  pseudoglobulin  portion.  They  are 
relatively  stable,  serum  containing  them  showing  a  loss  of  about  i  per  cent,  or 
less  of  its  receptor  content,  for  months  after  removal  from  the  body,  if  kept 
sterile  and  in  a  cool,  dark  place  (ice  box).  An  exposure  of  6o°C.  to  8o°C.  for 
J/2  hour  destroys  them. 

Receptors  of  the  second  order  include  the  agglutinins  and  precipitins  and 
these  are  more  complex  than  receptors  of  the  first  order  having  two  distinct 
parts,  one  part  which  unites  with  the  substance  to  be  agglutinated  and  another 
part  that  produces  the  agglutination.  Receptors  of  the  second  order  remain 
active  in  serum  for  months  after  removal  from  the  body  if  kept  cool  and  sterile. 
An  exposure  of  %  h°ur  to  7o°C.  permanently  destroys  receptors  of  the  second 
order. 

Receptors  of  the  third  order,  the  most  complex  of  all,  include  the  lysins  and 
have  been  previously  described  (page  267). 

DISCUSSION  OF  THEORIES  OF  IMMUNITY 

The  theories  of  Metchnikoff  and  of  Ehrlich  both  recognize  that  active  im- 
munity is  dependent  upon  cellular  activity,  and  exaltation  of  it  is  the  result  of 
stimulation  (irritation)  of  the  cells  by  the  poisonous  substance  against  which 
immunity  is  enhanced. 

The  recognizable  mechanism  of  infection  and  immunity  amply  substantiate 
this. 

Numerous  observations  indicate  that  the  mechanism  of  infection,  resistance 
and  immunity  is  in  some  cases  substantially  as  explained  by  Ehrlich;  in  others 


IMMUNITY  271 

; 

as  described  by  Metchnikoff  and  Wright,  but  in  many  instances  neither  of  these 
hypotheses  harmonize  with  the  facts. 

The  method  of  attack  of  different  groups  of  bacteria  varies,  the  results  of 
their  assaults  vary  and  the  consequent  defence  and  resistance  of  the  host  vary 
to  combat  these  dissimilar  infections. 

Thus  we  observe  in  diphtheria  no  invasion  by  the  bacteria  but  intoxication 
with  extracellular  toxin  and  this  is  combated  largely  if  not  entirely  by  anti- 
toxin. 

In  typhoid  and  paratyphoid  infections,  leucocytosis  is  conspicuous  by  its 
absence;  convalescent  and  immune  persons  having  agglutinins,  precipitins  and 
lysins  in  their  blood  serum  in  greater  amount  than  before,  in  greater  amount 
than  is  generally  observed  in  the  serum  of  persons  not  immune  to  typhoid 
infection. 

In  acute,  localized  staphylococcus  and  streptococcus  infections,  leucocyto- 
sis and  phagocytosis  is  marked;  the  convalescent  patient's  serum  contains 
practically  no  antitoxin,  little  if  any  precipitin,  agglutinin  or  lysin,  but  does 
contain  an  increased  amount  of  opsonin. 

After  infection  or  treatment  with  dead  bacteria  or  bacterial  products  the 
result  is  not  always  immunity  or  exalted  resistance.  Some  species  of  bacteria 
may  infect  one  time  after  time  apparently  leaving  the  host.more  susceptible 
to  subsequent  infection  than  he  was  primarily,  suggesting  that  cellular  reaction 
as  described  by  Ehrlich  and  Metchnikoff  does  not  always  occur  and  that  recovery 
is  possible  without  it. 

ANAPHYLAXIS 

Metchnikoff  observed  in  unicellular  organisms  that  phagocytosis  was  not 
confined  to  invading  parasites  and  food;  inanimate  poisons  frequently  being 
disposed  of  in  the  same  way.  Also  in  the  study  of  agglutinin,  precipitin  and 
lysin  production,  Ehrlich  and  his  co-workers  discovered  that  such  antibodies 
were  generated  by  the  injection  of  many  inanimate  proteins. 

Using  the  term  antigen  (haptin)  to  designate  a  substance  which  when  intro- 
duced into  an  animal  is  a  foreign  irritant,  or  splits  into  simpler  compounds  some 
of  which  are  irritant  or  toxic,  and  stimulates  the  cells  of  the  injected  animal  to 
produce  specific  agglutinins,  precipitins,  antitoxins,  lysins,  or  other  antibodies 
or  specific  ferments,  and  as  a  result  makes  the  injected  animal  either  immune  or 
hypersusceptible  to  subsequent  injections,  we  find  a  great  variety  of  proteins 
in  common  animal  and  vegetable  matter  and  in  foods,  act  as  antigens. 

As  to  why  the  injection  of  dead  typhoid  bacilli  (antigen)  or  infection  with 
living  typhoid  bacilli  (antigen)  immunizes  against  .future  invasion  by  typhoid 
bacilli,  and  injection  or  ingestion  of  red  blood  cells  from  another  species  (anti- 
gen) or  blood  serum  from  another  species  (antigen)  or  egg  albumin  (antigen) 
creates  a  hypersensitiveness  to  the  injurious  effect  of  similar  subsequent  injections 
of  these  substances  is  a  matter  of  speculation. 

This  sensitization  by  foreign  proteins  is  a  serious  phenomenon  of  common 
occurrence. 

When  produced  by  bacteria  or  bacterial  products  it  is  referred  to  as  allergy. 


272  MEDICAL  BACTERIOLOGY 

When  produced  by  other  substances  it  is  referred  to  as  anaphylaxis. 

As  commonly  thought  of,  allergy  or  anaphylaxis  is  a  state  of  hypersensitive- 
ness  to  a  foreign  protein,  produced  by  the  primary  dose  of  that  protein  and 
manifest  only  after  a  second  dose. 

This  phenomenon  was  first  observed  by  Theobald  Smith  when  studying  the 
effect  of  repeated  subcutaneous  injections  of  horse  serum  into  guinea-pigs.  He 
found  that  in  practically  all  cases  the  first  injection  of  horse  serum  into  a  guinea- 
pig  is  innocuous;  the  animal  shows  no  subsequent  discomfort,  illness  or  injury. 
A  second  injection,  somewhat  less  in  quantity,  equal  to,  or  greater  than  the 
first,  in  a  few  minutes,  always  less  than  10,  and  sometimes  almost  instantly, 
produces  signs  of  great  distress  and  usually  death. 

The  animal  shows  difficulty  in  breathing,  air  hunger,  throws  up  its  head 
falls  paralyzed  and  expires.  Occasionally  the  animal  does  not  die  but  lies  on  its 
side  breathing  rapidly  for  several  minutes  or  hours  and  then  quickly  recovers 
completely. 

Guinea-pigs  that  have  manifested  these  signs  of  hypersensitiveness  and  have 
recovered  are  thereafter  non-sensitive,  a  subsequent  injection  or  injections  of 
horse  serum  causes  no  illness  or  injury — the  animals  are  immune. 

The  state  of  hypersensitiveness  does  not  develop  immediately  after  the  first 
injection;  it  develops  gradually,  does  not  reach  its  fastigium  until  2  or  3 
weeks  after  the  first  injection  and  may  be  deferred.  There  is  a  minimum  quan- 
tity of  horse  serum  which  will  produce  anaphylaxis  and  larger  quantities  having 
the  same  effect.  The  larger  the  quantity  used  for  the  first  injection  the  longer 
the  interval  before  hypersensitiveness  develops. 

During  the  period  of  incubation — the  interim  between  the  first  injection  and 
the  development  of  hypersensitiveness — a  second  injection  causes  no  disturbance, 
but  somewhat  lengthens  the  period  of  incubation  beyond  what  it  otherwise 
would  be.  Multiple  injections  during  the  period  of  incubation  have  the  same 
effect.  A  guinea-pig  sensitized  to  horse  serum  continues  hypersensitive  to  it 
throughout  life  unless  immunized,  so  that  a  second  injection  given  a  year  after 
the  first  will  be  followed  by  a  typical  anaphylactic  attack. 

As  small  a  quantity  as  i/iooo  cc.  of  horse  serum  subcutaneously  has  caused 
a  fatal  reaction  in  sensitized  guinea-pigs  and  1/50,000,000  cc.  of  pollen  extract 
has  induced  a  violent  attack  of  hay  fever. 

Many  investigators  throughout  the  world  have  confirmed  the  observations 
of  Smith. 

Recent  studies  have  shown  that  anaphylaxis,  like  immunity,  may  be  pas- 
sive or  active,  passed  from  mother  to  progeny  or  acquired,  and  may  be  due  to 
the  blood  serum  alone,  fixed  tissue  cells  alone  or  both. 

The  phenomena  of  anaphylaxis  and  allergy — sensitization  by  a  primary  non- 
toxic  dose  after  a  period  of  incubation,  sudden  onset  of  respiratory  disturbance 
and  paralysis  following  a  second  injection  and  terminating  in  death  or  complete 
recovery  with  subsequent  immunity — -has  been  produced  in  guinea-pigs  and 
other  animals  with  extract  of  oats,  egg  albumin,  other  proteins  and  bacteria. 

From  these  observations  and  certain  similarities  to  what  has  been  well 


IMMUNITY  273 

known  as  idiosyncrasy,  it  has  been  assumed  by  some  that  anaphylaxis,  allergy 
and  idiosyncrasy  are  essentially  the  same.  This  may  or  may  not  be  true. 

There  are  two  apparent  differences  between  idiosyncrasy  and  allergy  or 
anaphylaxis:  idiosyncrasy  is  a  hypersensitiveness  disclosed  by  a  primary  dose 
of  the  irritant;  this  hypersensitiveness  persists  after  recovery  from  one  or  more 
doses  that  precipitate  symptoms  of  intoxication. 

Those  who  hold  the  opinion  that  anaphylaxis,  allergy  and  idiosyncrasy  are 
basically  the  same,  look  upon  luetin,  mallein,  typhoidin  and  tuberculin  reactions 
as  anaphylactic  manifestations.  Until  more  is  known  it  is  unsafe  to  generalize. 

Sensitization  to  tuberculin  may  be  general  or  localized.  An  animal  afflicted 
with  active  tuberculosis  causing  general  toxemia  shows  a  generalized  hypersen- 
sitiveness to  tuberculin;  should  the  disease  become  dormant,  hypersensitive- 
ness  may  continue  at  the  same  degree  as  when  the  disease  was  active  or  it  may 
be  reduced;  or,  general  sensitization  may  decline,  but  certain  organs  or  tissues, 
as  the  skin  and  conjunctiva,  continue  equally  as  sensitive  in  the  dormant  state 
of  the  disease  as  they  were  in  the  active  state. 

Local  sensitization  to  tuberculin  without  generalized  sensitization  can  be 
produced  in  non-tuberculous  animals.  The  instillation  of  tuberculin  will 
sensitize  the  conjunctiva  of  a  healthy  rabbit's  or  man's  eye  so  that  a  second 
instillation  causes  a  violent  local  reaction,  while  a  primary  instillation  in  the 
opposite  eye,  at  the  time  of  the  second  instillation  in  the  sensitized  eye,  pro- 
duces no  reaction. 

The  recent  investigations  of  Manwaring  and  Kusama  show  that  in  some 
instances,  at  least,  a  dose  of  antigen  administered  to  a  sensitized  animal  im- 
mediately acts  on  the  lungs  in  a  manner  that  greatly  impairs  or  totally  prevents 
respiration  and  aeration  of  the  blood.  While  such  pathological  changes  account 
for  the  respiratory  distress  commonly  observed  in  anaphylactic  attacks  and 
could  be  responsible  for  sudden  death,  they  do  not  explain  the  paralysis.  Other 
investigators  have  shown  involvement  of  other  vital  organs,  especially  the 
kidneys. 

By  perfusing  the  lung  of  a  normal  non-sensitized  animal,  with  a  mixture 
of  blood  serum  and  antigen  from  a  sensitized  animal,  the  same  pathological 
changes  are  produced  as  occur  in  a  sensitized  animal  following  a  dose  of  antigen. 

Manwaring  and  Kusama  state  that  the  serum  of  a  guinea-pig  immune  to 
horse  serum,  when  mixed  with  horse  serum  makes  it  inert  just  as  diphtheria 
antitoxin  neutralizes  diphtheria  toxin,  so  that  the  injection  of  a  mixture  of 
immune  guinea-pig  serum  and  horse  serum  into  a  sensitized  guinea-pig  does  not 
produce  any  manifestation  of  anaphylaxis.  They  also  report  that  a  guinea-pig 
immunized  to  horse  serum  enjoys  immunity  by  virtue  of  substances  in  its  blood 
serum  which  act  on  horse  serum  and  neutralize  it  before  it  reaches  fixed  tissue 
cells.  The  fixed  tissue  cells  of  such  immune  animals  are  hypersensitive  to 
horse  serum;  this  being  shown  by  freezing  the  lungs  of  an  immune  guinea-pig 
of  their  native  blood  and  perfusing  them  with  a  mixture  of  normal  guinea- 
pig  serum  and  horse  serum,  the  perfusion  causing  a  typical  anaphylactic 
manifestation. 

18 


274  MEDICAL  BACTERIOLOGY 

It  is  a  natural  and  common  tendency  to  explain  anaphylaxis  so  as  to  har- 
monize it  with  Ehrlich's  theory  of  immunity:  assuming  that  the  first  injection 
or  ingestion  of  antigen  does  not  produce  ill  effect  because  it  is  not  toxic  as  taken 
in,  only  becoming  toxic  when  disintegrated;  at  the  time  the  body  receives  its 
first  dose  of  antigen  the  body  cells  and  fluids  are  poor  in  ferments  or  receptors 
or  both,  capable  of  disintegrating  the  antigen,  hence  it  is  slowly  disintegrated, 
the  toxic  substance  being  slowly  liberated  in  quantities  too  small  to  irritate; 
this  antigen  must  be  disintegrated  (to  feed  the  body  or  be  eliminated)  and  its 
presence  is  a  stimulus  to  the  body  cells  to  produce  much  more  ferment  and 
receptors  capable  of  disintegrating  antigen  and  hence  capable  of  liberating  its 
toxic  components;  this  stimulated  new  activity  of  the  body  cells  results  in  a 
gradual  increase  of  ferments  and  receptors,  the  quantity  necessary  to  disinte- 
grate antigen  so  rapidly  as  to  liberate  at  once  sufficient  toxin  to  produce  obvious 
injury,  doe?  not  develop  or  accumulate  until  one  or  more  weeks  after  the  stimulus 
to  production  caused  by  the  first  dose  of  antigen;  a  second  injection  of  antigen 
given  in  the  early  days  of  increased  antibody  production  (period  of  incubation) 
does  not  produce  ill  effect  for  the  same  reason  that  the  primary  does  not;  a 
second  injection  of  antigen  given  when  antibody  production  has  reached  its 
maximum  (period  of  hypersensitiveness)  produces  ill  effect  because  it  is  rapidly 
disintegrated  and  therefore  an  injurious  quantity  of  toxin  suddenly  liberated; 
animals  that  survive  this  injury,  by  it  have  their  cells  stimulated  to  form  recep- 
tors or  ferments  for  the  toxic  component  liberated  when  antigen  is  disintegrated, 
hence  subsequent  doses  of  antigen  do  not  produce  ill  effect  (the  animal  has  lost 
its  hypersensitiveness,  is  immune)  because  there  are  antibodies  in  the  animal's 
blood  serum  that  neutralize  the  toxin  as  fast  as  it  is  liberated  by  disintegration 
of  antigen.  This  explanation  is  largely  hypothetical. 

RELATIONSHIP  OF  ALLERGY,  ANAPHYLAXIS  AND  IDIOSYNCRASY  TO  IMMUNITY 

In  a  previous  chapter  immunity  was  described  as  a  relative,  not  absolute, 
state  subject  to  fluctuations;  equally  dependent  upon  intrinsic  and  extrinsic 
factors. 

Susceptibility  and  hypersusceptibility  (hypersensitiveness)  are  also  relative 
conditions  subject  to  changes  and  dependent  upon  both  intrinsic  and  extrinsic 
factors.  Furthermore,  it  is  observed  that  in  many  instances  the  same  factors 
that  are  active  in  producing  immunity  are  also  active  in  producing  allergy  and 
anaphylaxis.  All  the  demonstrable  facts  relative  to  these  conditions  indicate 
that  they  are  chemical  reactions  fundamentally  related. 

It  is  well  known  that  living  organisms  in  general  try  at  times  with  a  sur- 
prising degree  of  success  and  again  with  conspicuous  failure  to  cope  with  changes 
in  their  environment  and  pabulum;  of  two  men  transferred  from  a  balanced 
diet  to  a  deficient  diet  one  will  adjust  himself  to  the  change  and  continue  to 
enjoy  good  health,  the  other  fails  to  and  suffers  scurvy  or  beriberi;  of  two  patho- 
genic bacteria  invading  a  man  under  identical  conditions,  one  fails  to  withstand 
the  unfavorable  elements  of  its  environment  and  dies,  the  other  survives,  mul- 
tiplies and  produces  infection.  In  such  cases  vulnerability  depends  upon  the 


IMMUNITY  275 

lack  of  power  to  increase  or  change  the  chemical  activity  of  certain  cells  and 
fluids  of  the  body;  immunity  results  from  the  possession  of  such  power. 

It  would,  therefore,  seem  that  idiosyncrasy  is  a  manifestation  of  cellular 
inability  to  respond  to  a  need  for  increased  or  new  chemical  activity,  that 
allergy  and  anaphylaxis  are  manifestations  of  imperfect,  inadequate  response 
and  that  active  immunity  is  the  result  of  adequate  response  to  a  need  for  greater 
or  more  diversified  chemical  activity  of  certain  tissue  cells. 

Much  is  yet  to  be  learned  as  to  which  cells  produce  antibodies,  variations  in 
their  response  to  different  antigens  and  the  exact  chemical  nature  of  various 
antigen-antibody  reactions.  The  application  to  these  problems  of  better  meth- 
ods of  studying  colloidal  changes  and  hydrogen  ion  concentration  will  extend 
our  knowledge  of  the  mechanism  of  immunity  and  variations  in  response  of  the 
host  to  antigens. 

Although  the  extensive  studies  of  Victor  Vaughan  and  Abderhalden  have 
not  produced  any  new  technique  or  working  hypothesis  that  at  present  can  be 
applied  in  medical  practice,  they  have  disclosed  many  facts  relative  to  the 
chemical  aspect  of  bacterial  life  and  the  presence  of  ferments  in  blood  serum 
that  every  immunologist  and  physician  should  become  familiar  with. 

ANIMAL  INOCULATION 

Animal  inoculations  are  occasionally  helpful  in  establishing  a  diagnosis 
when  other  means  of  investigation  fail  or  are  inadequate;  they  are  indispensable 
in  attempts  to  detect  and  identify  the  offending  organism  in  infectious  diseases 
of  unknown  origin  and  in  the  development  of  specific  chemical  preparations  for 
the  alleviation,  cure  and  prevention  of  diseases  that  afflict  men  and  brutes;  they 
are  essential  to  the  production  of  specific  sera  that  in  diagnosis  and  treatment 
curtail  immeasurably  the  sufferings  of  man  and  beast. 

If  one  considers  the  sheep  that  have  been  saved  from  anthrax,  the  hogs 
saved  from  cholera,  the  horses  saved  from  lockjaw  and  animals  of  all  sorts  saved 
from  hydrophobia,  as  a  direct  result  of  animal  inoculations,  and  compares  this 
with  the  total  suffering  inflicted  upon  dumb  animals  by  man  in  his  effort  to 
arrest  disease,  the  conviction  that  these  activities  have  lessened  the  sufferings 
of  brutes  is  unavoidable. 

Before  one  can  procure  desired  results  from  animal  inoculation  tests  in  the 
study  of  bacteriology  several  things  are  necessary — familiarity  with  the  appear- 
ance and  habits  of  experimental  animals  in  health,  the  effect  of  sudden  changes 
of  environment  upon  them,  their  normal  rate  of  growth,  the  normal  appearance 
and  relations  of  their  internal  organs,  the  diseases  that  occur  spontaneously  in 
these  animals  and  the  changes  they  produce,  and  finally,  the  chain  of  events  to 
be  expected  when  an  animal  has  been  inoculated  with  a  particular  species  of 
pathogenic  bacteria. 

Postmortem  examinations  of  experimental  animals  should  be  made 
and  recorded  with  the  same  care  and  detail  and  by  the  same  general  tech- 
nique described  in  text  books  on  human  pathology  in  which  direction  will 
also  be  found  for  the  removal,  fixation  and  staining  of  tissue  for  microscopic 
examination. 


INDEX  TO  SUBJECTS 


Achorion  Schonleini,  159 

Acid,  formation  by  bacteria,  129 

Acids,  as  germicidal  agents,  24 

Acne,  201 

Acquired  Immunity.     See  Immunity. 

definition  of,  262 
Actinomyces,  8,  204 

cultivation  of,  156,  157,  158 

morphology  of,  156 

occurrence  of,  155,  157 

staining  of,  156 

Active  immunity.     See  Immunity. 
Acute  anterior  poliomyelitis,  169 
Aerobe,  6,  53 
Aerobic  organisms,  obligatory,  6 

facultative,  6 
Agar  for  culture  media,  43 

blood,  45 

blood  smeared,  45 

slants,  for  cultivation  of  bacteria,  50 
Agglutination  reactions,  65,  66,  67,  in,  112, 

H3 

clinical  diagnosis  by,  in  typhoid,  in 
in    differentiating    various    types    of 
pneumococci,  65,  66,  67 

differentiation  of  various  bacterial  spe- 
cies by,  65,  66,  67,  113 

group  agglutination  in,  113 

macroscopic  observation  of,  for  deter- 
mination of  titer  limit,  112 

macroscopic  observation  of,  for  clinical 
diagnosis,  in,  112 

specificity  of,  113 

upon  dead  bacteria,  112 

upon  living  bacteria,  1 1 1 
Agglutinins,  267 

diluted,  agglutination  reaction  with,  112- 
114 

experimentation  with,  65-113 

group,  113 

in  agglutination  reactions,  111 

in  various  immune  sera,  1 1 1 

nature  of   113 

production  of,  in  sera  of  animals,  by,  65 
injection  of  bacteria,  113 

reaction  of,  113 

specificity  of,  113 


Aggressins,  259 

action  of,  259 

character  of,  259 

occurrence  of,  259 

theoretical  consideration  of,  259 
Air,  bacteria  in.     See  Individual  organisms. 
Alcohol,  28 

antiseptic  properties  of,  28 

as  fixative  in  staining,  22 

fermentation  by  yeasts,  164 

production  of,  164 
Alkalies,  as  germicidal  agents,  24 
Allergy,  271,  274 
Amboceptor,  234,  235,  236,  237 

consideration  of,  234,  235 

determination  of  in  Wassermann   test, 

237 

fixation  of  complement  by,  234 
occurrence     and     properties    of,     234, 

237 

Anaerobe,  6,  53   54 
Anaerobic  bacteria,  6,  53,  54 

cultivation  of,  53,  54 

facultative,  6 

obligatory,  6 

occurrence  of,  130,  132,  134,  136 
Anaphylaxis,  271 

definition  of,  271 

experimentation  in,  271,  272,  273 

observations  in,  272 

phenomena  of,  271 

proteid  injection  in,  271 

symptoms  in,  272 

theoretical     consideration     concerning, 

273 

Anilin-gentian  violet,  18 
Anilin  water,  18 
Animals,  275 

experimentation,  275 

inoculations  in,  177,  275 
Anthrax,  139 

causes  of,  139,  140 

occurrence  of,  140 

vaccine,  222 
Anthrax   bacillus,    entire   consideration    of, 

139-140 
Antianthrax  serum,  225 


277 


278 


INDEX    OF    SUBJECTS 


Antibodies,  266,  267 

experimentation   and    facts   concerning, 
agglutinins,  65,  66,  67,  in,  112,  113 
amboceptors,  234,  235 
antitoxin,  223 
bacteriolysins,  267 
opsonins,  266 
precipitins,  267 
Anticomplementary,  249 
Antigen,  243,  247,  248,  249,  256,  257 
definition  of,  243 
fof    Wassermann    test,    243,    247,    248, 

249 
for  other  complement  fixation  tests,  256, 

257 

Antimeningococcus  serum,  225 

Antipneumococcus  serum,  67 

Antiseptic,  determining  strength  of  an,  185 

Antistreptococcic  sera,  224 

Antitoxin,  diphtheria.     See  Diphtheria  anti- 
toxin, 
tetanus.    See  Tetanus  antitoxin. 

Arnold  steam  sterilizer,  37 

Arthritis,  210 

Arthrospores,  consideration  of,  6 

Asiatic  cholera.     See  Cholera. 

Attenuation  of  cultures  in  manufacture  of 
vaccines,  39 

Autoclave,  details  of,  for  production  of  steam 

under  pressure,  35,  36,  38 
Trilatt,  30 

use  of,  in  generating  gaseous  formalde- 
hyde, 30,  31 

Avian  tuberculosis,  bacillus  of.     See  Tubercle 
bacillus. 

Bacilli,  classification  of,  4-7 

colon-typhoid  group  of,  103-118 
differentiation,    by    cultural    character- 
istics, 118 
by  morphology,  7 
Bacillus,  aerogenes  capsulatus,  132 
cultivation  of,  132 
morphology  of,  132 
occurrence  of,  132 
pathogenicity  of,  133 
resistance  of,  132 
staining  of,  132 

anthracis.     See  Anthrax  bacillus. 
Bordet-Gengou,  78 
botulinus,  130 
bulgaricus,  129 

coli  communis,  consideration  of,  103 
cultivation  of,  103 


Bacillus  coli  communis,   differentiation  from 

other  organisms,  118 
distribution  of,  103 
isolation  of,  on  special  media,  117 

(Plate) 

morphology  of,  103 
pathogenicity  of,  104 
staining  of,  103 

diphtheriae.     See  Diphtheria  bacillus, 
dysenteriae.     See  Dysentery  bacillus. 
Eberth  Gaffky.     See  Typhoid  bacillus, 
enteritidis,  characteristics  of,  no 
Friedlander.     See  Bacillus  mucosus  cap- 
sulatus. 
Hoffmani,  89 

influenzas.     See  Influenza  bacillus. 
Klebs-Loeffler.     See  Diphtheria  bacillus. 
Koch- Weeks,  78 
lactis  aerogenes,  129 
leprae.     See  Leprosy, 
maligni  oedematis,  134 
mallei.     See  Glanders, 
consideration  of,  147 
cultivation  of,  147 
morphology  of,  147 
occurrence  of,  147 
pathogenicity  of,  in  animals,  148 

in  man,  148 
staining  of,  147 
toxin  of.     See  Mallein. 
Morax  and  Axenfeld,  79 
mucosus  capsulatus, "association  of  with 

other  organisms,  8ij 
characteristics  of,  80 
cultural,  80 
morphological,  80 
pathological,  81 
staining  of,  80 
cedematis  maligni,  134 
paratyphoid,  characteristics  of,  109 
differentiation  of,   from  other  organ- 
isms, 118 

from  various  types,  118 
occurrence  of,  109 
pathogenicity  of,  109 
pestis.     See  Plague, 
pneumonias.     See  Pneumonia, 
proteus  vulgaris,  128 
prodigiosus,  143 
pseudo-diphtheria,  89 
pyocyaneus,  cultivation  of,  126 
morphology  of,  126 
occurrence  of,  126 
pathogenicity  of,  127 


INDEX    OF    SUBJECTS 


279 


Bacillus    pyocyaneus,   pigment    production 

of,  126 

staining  of,  126 
virulence  of,  127 

smegmatis.     See  Smegma  bacillus. 

subtilis,  141 

tetani.     See  Tetanus. 

tuberculosis,  avian,  bovine  and  human 
types  of.     See  Tubercle  bacillus. 

typhi-exanthematici,  169 

typhosus.     See  Typhoid. 

xerosis,  90 
Bacteria,  8 

acid  formation  by,  1 29 

acid  fast  stains  for,  19 

acid  and  alcohol  fast  stains  for,  19-20 

aerobic,  6 

agents    injurious    to.     See    Sterilization 
and  disinfection. 

anaerobic,  6,  53 

as  causes  of  disease,  263 

attenuated   or   dead   bacteria   used   for 
immunization.     See  Vaccine. 

biological  consideration  of.     See  Individ- 
ual organisms. 

chromogenic,  55,  126,  143 

classification  of,  4 
morphological,  4 
staining,  15,  19 

cultivation  of,  by  aerobic  methods,  50 
by  anaerobic  methods,  53,  54 

destruction    of.     See    Sterilization    and 
disinfection. 

discovery  of,  i 

differentiation  of,  by  cultural  methods,  5 1 
by  fermentation,  118,  119 

enzymes  produced  by,  259,  260  ' 

gas  formation  by,  52 

gram  negative,  16,  17 

gram  positive,  16,  17 

indol  production  by,  52 

isolation  of,  54 

life  of,  6 

liquefaction    of    gelatin    by.     See   Indi- 
vidual organisms. 

occurrence  of,  i 

parasitic,  definition  of,  7 

pathogenic,  definition  of,  7 

pigment  production  by,  55,  126,  143 

propagation  of,  by  fission,  4 

reproduction  of,  4 

saprophytic,  definition  of,  6 

spore  formation  of,  5,  6,  8 

staining  of,  19 


Bacteria,  structure  of,  5-8 

thermal  death  points  of,  39 

virulence  of,  260 
Bacteriaceae,  4 

Bacteriological  examination  of  blood  cultures, 
98,  114 

cat-gut,  179 

feces,  99,  116 

fluid,  178 

milk,  176 

quinine,  179 

sputum,  13,  97 

unknown  culture,  13,  178 

urine,  98-116 

water,  173 
Bacteriology,  development  and  early  history 

of,  i 

Bacteriolysins,  267 
Bacterium,  4 
Beggiatoa,  genus,  5 
Beggiatoaceae,  5 
Bichloride  of  mercury,  24,  33 
Bile  medium,  42 
Bismarck  brown,  19 
Blastomycetes.     See  Yeast. 
Blood  agar,  45 

media,  45 

serum  (Loeffler),  45 

staining  of,  2  r 

Bordet-Gengou  bacillus,  consideration  of,  78 
Botulism,  130 
Bouillon,  41 
Bovine  tuberculosis,  bacillus  of.    See  Tubercle 

bacillus. 

Brilliant  green,  as  an  antiseptic,  33 
Broth  used  for  culture  media,  41 

calcium  carbonate,  42 

glycerin,  41 

meat  extract,  41 

meat  infusion,  41 

sugar,  41 

Calcium  carbonate  broth,  42 
Calumette's  ophthalmo  test,  218 
Capsule,  staining  of,  in  bacteria,  21 
surrounding  of  bacteria  by  a,  5 
Carbol-fuchsin,  20 
Carbolic  acid,  as  disinfectant,  28,  33 
Carbolic  acid  coefficient,  185 
Carbuncles,  202 
Carrel-Dakin  solution,  25 
Cell  receptors,  various  forms  as  explained  in 

Ehrlich's  theory,  269 
Chancre,  examination  of,  152,  208 


2  &0 


INDEX    OF    SUBJECTS 


Chancre,  treponema  pallidum  in  a,  152 
Chlamydobacteriaceae,  4 
Chloramine  as  an  antiseptic,  28-33 
Chlorinated  lime  as  a  disinfectant,  25 
Chlorine  as  a  disinfectant,  25-33 
Cholera  asiaticae,  diagnosis  of,  123 

discovery  of,  2 

epidemics  of,  122 

findings  of,  123 

spirillum  of,  consideration  of,  122 
Chromogenic  bacteria,  55 
Cladothrices,  155 
Classification  of  bacteria,  4 
Clearing  of  media,  49 

Coagulation  of  milk  by  bacteria.     See  Indi- 
vidual organisms. 
Coccaceae,  4 
Cocci,  7 

Coley's  fluid,  221 

Colon  bacillus.  See  Bacillus  coli  communis. 
Colon-typhoid,  differentiation,  media  for,  119 
Colonies,  counting  of,  on  plates  of  media,  53, 

175 
Comma    bacillus.     See     Spirillum    choleras 

asiaticae. 
Complement,  consideration  of,  234,  238 

definition  of,  234 

standardization  of,  for  use  in  Wasser- 

mann  test,  238 
Complement  fixation,  action  in,  226 

in  serum  reactions,  226 

in  various  diseases,  256 

Wassermann  test  for,  226 
Conjunctiva,  205 
Conradi-Drigalski  medium,  44 
Counting  of  bacteria,  175 
Cover-glasses,  n 
Crenbthrix,  5 
Cultivation  of  bacteria,  aerobically,  50-53 

anaerobically,  53,  54 
Culture  media,  40-54 

filtration  of,  48 

formulas,  41 

technique  and  mode  of  preparation  and 
sterilization  of  all,  41-46 

titration  of,  for  standard  work,  47,  48 
Culture  technique,  51 

Dakin-Carrel  solution,  25 

Death  point,  thermal,  for  bacteria,  39 

Destruction  of  bacteria,  by  chemicals,  24-33 

by  physical  agents,  35-39 
Differential  staining  for  bacteria.     See  under 
Staining 


Diphtheria  antitoxin,  discovery  of,  2 
production  of,  223 
pseudo-globulins  of,  223 
standardization  of,  223 
unit  of,  224 

Diphtheria  bacillus,  bacillus  similar  to,  90 
biological  consideration  of,  82,  83,  84 
cultivation  of,  84 
discovery  of,  2 

examination  for,  in  mouth,  86,  87 
occurrence  of,  82 
pathogenicity  of,  85 
resistance  of,  84 
staining  of,  82 
toxin,  properties  of,  85 
varieties  of,  83,  84 
Diplococci,  7 

diplococcus  lanceolatus.     See  Diplococ- 

cus  pneumonias, 
diplococcus  pneumonias,  63 
characteristics  of,  63 
cultivation  of,  64 
differentiation  of,  from  streptococcus, 

64 

morphology  of,  63 
pathogenicity  of,  64 
resistance  of,  64 
staining  of,  64 
types  of,  65 

Diseases  of  unknown  causation,  169 
Disinfectants,  chemical,  23 
gaseous,  30 
physical,  35 

Disinfection,  technique  of.     See  Sterilization. 
Dorsett  egg  medium,  45 
Ducrey's  bacillus,  208 
Dysentery  bacilli,  biological  consideration  of, 

119 

characteristics  of,  119 
morphology  of,  119 
pathogenicity  of  various  types  of,  1 20 
products  of,  1 20 
varieties  of,  119 

Ear,  diseases  of  the,  205 
Eberth,  discovery  of  typhoid,  2 
Edema,  malignant,  bacillus  of.     See  Malig- 
nant bacillus. 
Egg,  for  preparation  of  medium,  45 

for  use  in  culture  media,  49 
Eggs,  technique  for  examination  of,  183 
Ehrlich's  immunity  theory,  269 
Endo's  medium,  43 

for  colon-typhoid  differentiation,  117 


INDEX    OF    SUBJECTS 


28l 


Endospores,  8 
Endotoxins,  259 
Enzymes,  259,  260 

Eosin,  preparation  of  aqueous  solution  of,  16 
Erysipelas,  202.   Also  see  under  Streptococcus. 
Ethylhydrocuprein  hydrochloride,  33 
Eusol,  solution  of,  as  an  antiseptic,  28,  33 
Eye,  diseases  of  the,  264 

Facultative  aerobes,  6 

anaerobes,  6 
Farcy.    See  Glanders. 
Feces,  bacteriological  examination  of,  116 

disinfection  of,  25 

Fermentation  tests,  for  colon-typhoid  differ- 
entiation, 118 

in  water,  1 74 
Fish  tuberculosis,  bacillus  of.     See  Tubercle 

bacillus. 

Fixation  of  complement,  227 
Flagella,  special  staining  methods,  20 

structure  of,  5 

Flavine,  as  an  antiseptic,  29,  33 
Formaldehyde,  as  a  disinfectant,  29 

entire  consideration  of  all  common  meth- 
ods in  vogue  for  the  generation  of 
gaseous  solution  of,  30 
Fractional  sterilization,  36 
Frambcesia,  203 
Friedlander  bacillus.     See  Bacillus  mucosus 

capsulatus. 
Fuchsin,  15 

Fungi,  mould.     See  under  Hyphomycetes. 
Furunculosis,  202 
Fusiform  bacilli,  of  Vincent's  angina,  91 

Gabbet's  stain  for  tubercle  bacillus,  19,  20 

Gaertner,  discovery  of  bacillus  enteritidis,  no 

Gangrene,  171 

Gas  formed  by  bacteria,  52 

Gelatin  medium,  43 

liquefaction    of,    by   bacteria.     See   In- 
dividual organisms. 
Gentian-violet,  16 

Glanders,  bacteriological  diagnosis  of,   148, 
202 

bacillus.     See  Bacillus  mallei. 
Glycerin,  use  of,  in  media,  41 
Gonococcus,  complement  fixation  test  of  the, 
256 

cultivation  of,  71 

examination  for,  72 

infection  of,  in  man,  73 

morphology  of,  71 


Gonococcus,  pathogenicity  of,  72 

resistance  of,  72 

staining  of,  71 

vaccine  of,  212 
Gonorrhea,  73 

Gram  negative  bacteria,  16,  17 
Gram  positive  bacteria,  16,  17 
Gram's  method  of  staining  and  differentia- 
tion of  bacteria  by,  16 
Group  agglutination,  113 

Haffkine's  vaccine,  222 
Halogens  as  disinfectants,  25 
"Hanging  drop,"  method  of  preparation,  13 
Heat,  different  kinds  of,  in  use  for  steriliza- 
tion purposes,  35 
Hemolysis,  240 
Higher  bacteria,  the,  155 
Hiss'  serum  medium,  42 
Hydrophobia.     See  Rabies. 
Hypersusceptibility.     See  Anaphylaxis. 
Hyphomycetes,  classification  of,  162 

conditions  favorable  to  growth,  161 

diseases  caused  by,  favus,  159 
ringworm,  159 

morphology,  161 

varieties  of,  162 

Immunity,  acquired,  262 

definition  of,  259 

Ehrlich's  theory  of,  269 

Metchnikoff  theory,  268 

natural,  264 

phagocytic  theory  of,  265 

side-chain  theory  of,  269 
Impetigo  contagiosa,  203 
Indol,  production  of,  52 

tests  for,  52 

Infection,  consideration  and  definition  of,  263 
Influenza  bacillus,  77 

bacteria  related  to,  78 

biology  of,  77 

isolation  of,  77 

morphology  of,  77 

pathogenicity  of,  77 

staining  of,  77 
Inoculation  of  media,  51 
lodoform  as  disinfectant,  29 
Iodine    and    iodine    tetrachloride    as    disin- 
fectants, 25,  33 
Isolation  of  bacteria,  54 

Jenner's     discovery     of     immunization     in 
smallpox,  i,  171 


282 


INDEX    OF    SUBJECTS 


Ketchup,  examination  of,  179 

Klebs  bacillus.     See  Diphtheria  bacillus. 

Koch's  old  tuberculin,  214 

Kock- Weeks  bacillus,  78 

Lactose  media,  41 

agar,  41 

bouillon,  41 

litmus  agar,  41 
Leprosy,  204 

bacillus  of,  101 

cultivation  of,  101 

morphology  of,  101 

occurrence  of,  101 

pathogenici ty  of,  101 

relation  of,  to  tubercle  bacillus,  101 

staining  of,  101 
Leucocytes  in  milk,  177 
Light,  effect  of,  on  tubercle  bacillus,  96 
Lime,  chloride  of,  as  a  disinfectant,  25 
Litmus  milk,  41 
Lobar  pneumonia,  65 
Loeffler's  blood  serum  media,  45 

methylene  blue,  15 
Lungs,  diseases  of  the,  206 
Lupus,  203 
Lysins,  267 
Lysol,  29 

MacConkey's  bile  salt  media,  42 

Madura  foot,  204 

Malachite  green  as  an  antiseptic,  33 

media,  44 
Malignant  edema,  bacillus  of,  134. 

consideration  of,  134 

cultivation  of,  134 

morphology  of,  134 

pathogenicity  of,  134 

staining  of,  134 
Mallein,  148 
Malta  fever,  125 
Mastoiditis,  206 
Measles,  171 
Meat  extract  bouillon,  41 

preparation  of,  41 
Meat  infusion  bouillon,  41 

preparation  of,  41 

Meningitis,  microorganisms  causing,  68 
Meningococcus,  68 

cultivation  of,  68 

early  observation  of,  68 

morphology  of,  68 

pathogenicity  of,  69 

serum,  70 


Meningococcus,  viability  of,  69 

Metacresol  as  a  disinfectant,  28 

Methylene  blue,  Loeffler's,  15 

Micrococci,  7 

Micrococcus  catarrhalis,  consideration  of,  74 

intracellularis,   meningitidis.     See  Men- 
ingococcus. 

melitensis,  consideration  of,  125 

tetragenus,  consideration  of,  75 
Microorganisms,  classification  of,  4 

discovery  of,  i 

parasitic,  7 

pathogenic,  7 

saprophytic,  7 
Microscope,  dark-field,  n 

early  observations,  9 

present  study  of  bacteria  byxaid  of  the,  9, 

10,  ii 
Microsporon  audouini,  159 

furfur,  150 

Migula's  classification  of  bacteria,  4 
Milk,  animal  inoculation  tests  in,  177 

as  a  culture  media,  41 

bacteria  in,  176 

bacteriological  examination  of,  176 

diseases  caused  by,  176 

leucocytes  in,  177 

plating  test  in  examination  of,  176 
Monilia,  167 
Moro  test,  218 
Motility  of  bacteria,  4-8 
Moulds.     See  Hyphomycetes. 
Mucor,  162 
Mycomycetes,  162 

Negri  bodies,  in  rabies,  219 
Neisser's  method  of  staining  diphtheria  bacil- 
lus, 18 

" New  Tuberculin "  (Koch),  214 
"New  Tuberculin,"  bacillary  emulsion,  214 
Nose,  diseases  of  the,  206 

Obligatory  aerobes,  6 

anaerobes,  6 

"Old  Tuberculin"  (Koch),  214 
Opsonic  index,  2,  265 

test,  Wright's,  entire  consideration  of,  265 
Opsonins,  264 

Orthocresol,  as  a  disinfectant,  28 
Otitis  media,    205 
Oxygen,  as  a  disinfectant,  32 

Pappenheim's  solution,  in    use  for  staining 
tubercle  bacillus,  20 


INDEX    OF    SUBJECTS 


Paracresol,  as  a  disinfectant,  28 

Parasites,  bacterial,  7 

Paratyphoid  bacillus,  109 

Passive  immunity.     See  Immunity. 

Pathogenic  bacteria,  7 

Penicillium,  162 

Peroxide  of  hydrogen,  as  a  disinfectant,  24 

Petri  dish  plates,  50 

PetrofFs  media,  46 

modifications  of,  46 
Pfeiffer's  phenomenon,  123,  264 
Phagolysis,  264 

Phenol,  as  a  disinfectant,  28,  33 
Phenol  coefficient  test,  185 
Physiological  normal  salt  solution,  227 
Pigment  formation  by  bacteria,  55,  126,  143 
Plague,  bacillus  of,  biology  of,  144 

cultivation  of,  145 

morphology  of,  144 

occurrence  of,  144 

pathogenicity  of,  145 

resistance  of,  145 

staining  of,  144 
Planococcus,  4 
Planosarcina,  4 
Plating,  52 
Pneumobacillus.     See  Bacillus  mucosus  cap- 

sulatus. 

Pneumococcus.    See  Diplococcus  pneumonias. 
Poisons,  bacterial.     See  Enzymes. 
Polar  bodies,  special  stains  for.     See  Neis- 

ser's  method,  under  staining. 
Poliomyelitis,  acute  anterior,  169 
Potassium  dichromate,  as  a  disinfectant,  24 

permanganate,  as  a  disinfectant,  24 
Potato  media,  46 
Precipitins,  267 

Preparation  of  specimens  for  examination,  1 1 
Pseudo-diphtheria  bacillus,  89 
Purpura  hemorrhagica,  203 
Pus,  bacteriological  examination  of,  118 
Pus  cells  and  leucocytes  in  milk,  117 
Pyocyanin,  126 

Pyrogallic    acid,    use    of,    in    cultivation    of 
anaerobic  bacteria,  53,  54 

Rabies,  diagnosis  of,  218 

negri  bodies  in  central  nervous  system  in 

cases  of,  219 
occurrence  of,  218 
pathology  of,  219 
treatment   of   cases   with   injections   of 

attenuated  virus  fixe,  220 
virulence  and  preparation  of  virus,  220 


Receptors,  269 

Red  blood  cells,  for  Wassermann  and  other 

complement  fixation  tests,  227 
Reducing  power  of  bacteria,  52 
Relapsing  fever,  spirochaeta,  145 
Reproduction  of  bacteria,  4 
Ringworm,  159 

Saccharose  media,  41 

Saccharomycetes.        See   Yeasts   and   yeast 

cells. 

Salt,  normal  physiological,  solution,  227 
Saprophytes,  6 
Saprophytic,  definition  of,  6 
Sarcina,  4,  7,  75 
Scarlet  fever,  171 
Schizomycetes,  8 

Sclavo's  serum.     See  Antianthrax  serum. 
Sera,  therapeutic,  223-225 
Serum  media,  Hiss,  42 

LoefHer's  blood,  45 
Serum  reactions,  technique  of,   agglutination 

tests,  macroscopic,  in 
microscopic,  in 
Widal,  in 

complement  fixation,  226 
gonococcus,  256 
other,  256 
Wassermann,  226 
Schick  test,  88 
Side-chain  theory,  269 
Skin  diseases,  201 
Slides,  ii 
Smallpox,  etiological  factor  of,  171 

immunization  in,  171 

occurrence  of,  171 

treatment  of,  by  virus,  221 
Smegma  bacillus,  101 

cultivation  of,  101 

differentiation  from  tubercle  by  staining, 
102 

occurrence  of,  101 
Sodium  citrate  solution,  227 
Solutions,  stock,  for  staining  bacteria,  15 
Spinal  fluid,  examination  of.  70,  98,  249 
Spirilla,  8 
Spirillaceae,  4 
Spirillum,  description  of,  8 

cholerae  asiaticae.    See  Cholera. 

of  Vincent,  91 
Spirochaetae,  dentium,  154 

icterohemorrhaga,  170 

obermeyeri,  145 

of  relapsing  fever,  145 


284 


INDEX    OF    SUBJECTS 


Spirochaetae,  pallida,  cultivation,  151 

demonstration     of,     in     smears,     by 

Giemsa's  stain,  150 
in  tissue,  by  Levaditi's  method,  150 
morphology  of,  150 
occurrence  of,  in  syphilis,  150 
staining  of,  150,  151 
pertenius,  154 
refringens,  154 
Vincent's,  91 

Spores,  varieties  of,  5,  6,  8 
arthrospores,  6 
bacterial,  5,  6,  8 
endo  or  true,  8 
Sporotrichum  Schenkii,  168 
Sputum,  collection  of,  97 
examination  of,  97 
staining  in  examination  of,  97 
Staining,  15 

acid  fast  bacteria,  19 

and  alcohol  fast  bacteria,  19,  20 
blood  cell,  21 
Gabbet's  method  of,  for  tubercle  bacillus, 

19,  20 

Gram's  method  of,  16 
method  of,  for  capsules,  21 
flagella,  20 
spores,  21 

Neisser's   method,  for  diphtheria  bacil- 
lus, 18 
Pappenheim's    method   of,  for  tubercle 

bacillus,  19,  20 

preparation  of  solutions  for,  15-22 
Standardization  of,  amboceptor  for  Wasser- 

mann  test,  237 

antigen  for  Wassermann  test,  248 
antitoxin,  diphtheria,  224 

tetanus,  224 

complement  for  Wassermann  test,  238 
Staphylococci,  7,  55 
Staphylococcus,  55 

epidermis  albus,  55 
pyogenes  albus,  55 
aureus,  55 
citreus,  55 

cultural  characteristics,  56 
morphology  of,  55 
occurrence  of,  55 
pathogenicity  of,  57 
resistance  of,  57 
toxic  products  of,  57 
vaccine  of,  212 
virulence  of,  57 
Steam  in  sterilization,  36,  37,  38 


Sterilization,  23-39 

chemical,  23 

fractional,  36 

pharmaceutical,  32,  34,  35 

thorough  consideration  of,  23-39 
Sterilizer,  Arnold's  steam,  37 

hot  air,  35 

Streptococci,  4-7,  59,  170 
Streptococcus  pyogenes,  59 

classification,  59 

differentiation    from    other    organisms, 
61,  62 

morphology  of,  59 

occurrence  of,  59 

pathogenicity  of,  60 

resistance  of,  60 

staining  of,  59 

toxic  products  of,  60 
Streptothrices,  4,  155 
Streptothrix  madurae,  158 
Syphilis,  203 

microorganism  of.     See  Spirochaeta  pal- 
lida. 

Test,  complement  fixation,  226 
Wassermann,  226 
Widal,  in 
Tetanus  antitoxin,  224 

production  of,  224 
standardization  of,  224 
Unit  of,  224 

bacillus,  characteristics  of,  136 
cultivation  of,  136 
morphology  of,  136 
occurrence  of,  136 
pathogenicity  of,  137 
resistance  of,  137 
spores  of,  136 
toxin  of,  137 
Tetrads,  7 

Thermal  death  points,  39 
Titration  of  culture  media,  47,  48 
Torulae,  164 
Toxin,  extracellular,  260 

intracellular,  259 

Trichomycetes.     See  Chlamydobacteriaceae.. 
Trichophyton,  159 
Tricresol,  28 
Trillat  autoclave,  30 
Tubercle  bacillus,  avian,  99 
bovine,  99 
ichthic,  99 

organisms  related  to,  101,  102 
of  leprosy,  101 


INDEX    OF    SUBJECTS 


Tubercle    bacillus,    organisms    related    to, 

smegmatis,  101 
Tubercle  bacillus,  biological  consideration  of, 

94 

cultivation  of,  95 
examination  for,  20,  94,  95 
by  Gabbet's  method,  20 
by  Pappenheim's  method,  94 
by  Ziehl-Nielson  method,  95 
in  feces,  99 
in  milk,  99 
in  sputum,  97 
in  urine,  98 

method  of  staining  for  the,  20,  94,  95 
morphology  of,  94 
pathogenicity  of,  96 
resistance  of,  95 
staining  of,  94 
toxic  products  of,  96 
Tuberculin,  213 
Tuberculosis,  mode  of  infection,  94 

tuberculin  as   a   diagnostic   reagent   in, 

217,  218 

in  the  treatment  of,  217 
Typhoid    bacillus,    biological    consideration, 

106 

cultivation  of,  106 
differentiation  of,  from  other  organisms, 

117,  118 
discovery  of,  2 
examination  and  isolation  of,  114,  116, 

118 

in  blood  during  disease,  114 
in  feces,  116 
in  sputum,  118 

in  urine,  116  , 

morphology  of,  106 
pathogenicity  of,  107 
staining  of,  106 
toxic  products  of,  106 
vaccine  of,  212 
Typhoid  fever,  diagnosis  of,  by  agglutinins  in 

blood  serum,  in 
by  blood  culture,  114 
Widal  test,  in 

Unit,  for  amboceptor,  237 
antigen,  244 
antitoxin,  224 
complement,  238 
diphtheria  antitoxin,  224 


Unit,  tetanus  antitoxin,  224 

Urethritis,  208 

Urine,  bacteriological  examination  of,  98,  116 

Vaccine,  autogenous,  211 

polyvalent,  211 

sensitized,  213 

stock,  an 

definition  of,  211 
production  of,  211 
Vaginitis,  208 
Variola.     See  Smallpox. 
Vincent's  angina,  spirochaeta  of,  91 

cultivation  of,  91 

consideration  of,  91 
Virulence,  260 
Virus  fixe,  220 
Von  Pirquet  test,  218 
Von  Ruck's  tuberculus,  215 

Wassermann  test  for  the  diagnosis  of  syph- 
ilis, 226 

amboceptor,  234,  237 
antigen  for,  247 

standardization  of,  248 
complement  in,  234,  238 
controls  in  the,  244 
preparation  for,  239 
reading  of,  246 
technique  of,  245 
value  of,  250   ' 
Water,  bacteria  in,  173 

number  of  colonies  of,  1 73 
pathogenic,  cholera,  175 

typhoid,  1 06 

bacteriological  examination  of,  173-175 
Welch  bacillus,  132 

Wertheim's      media      for      cultivation     of 
gonococcus,  71 

Yaws,  154 

Yeast  cells,  consideration  of,  164 

cultivation  of,  164 

pathogenic,  165 

reproduction  by  budding,  164 

saprophytic,  164 

varieties  of,  165 
Yellow  fever,  171 

Ziehl-Nielson's  method  for  staining  tubercle 
bacillus,  95 


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